U.S. patent number 8,129,396 [Application Number 12/567,060] was granted by the patent office on 2012-03-06 for 2-[1h-benzimidazol-2(3h)-ylidene]-2-(pyrimidin-2-yl)acetamides and 2-[benzothiazol-2(3h)-ylidene]-2-(pyrimidin-2-yl)acetamides as kinase inhibitors.
This patent grant is currently assigned to Telik, Inc.. Invention is credited to Natalia Aurrecoechea, Paul P. Beroza, Komath V. Damodaran, Karen Y. Pontius, Louise Robinson, Reyna J. Simon, Truong Vu, Kevin T. Weber.
United States Patent |
8,129,396 |
Aurrecoechea , et
al. |
March 6, 2012 |
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides and
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides as
kinase inhibitors
Abstract
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides
and 2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides and
their salts are kinase inhibitors, useful in the treatment of
cancer.
Inventors: |
Aurrecoechea; Natalia (Oakland,
CA), Beroza; Paul P. (Belmont, CA), Damodaran; Komath
V. (Cupertino, CA), Pontius; Karen Y. (Aromas, CA),
Robinson; Louise (San Carlos, CA), Simon; Reyna J. (Los
Gatos, CA), Vu; Truong (San Martin, CA), Weber; Kevin
T. (Carmel, IN) |
Assignee: |
Telik, Inc. (Palo Alto,
CA)
|
Family
ID: |
41268298 |
Appl.
No.: |
12/567,060 |
Filed: |
September 25, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100081653 A1 |
Apr 1, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61100869 |
Sep 29, 2008 |
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Current U.S.
Class: |
514/256;
544/335 |
Current CPC
Class: |
C07D
417/06 (20130101); C07D 401/14 (20130101); A61P
35/04 (20180101); C07D 403/06 (20130101); C07D
403/14 (20130101); A61P 35/02 (20180101); A61P
43/00 (20180101); A61P 35/00 (20180101); C07D
417/14 (20130101) |
Current International
Class: |
C07D
239/42 (20060101); C07D 401/04 (20060101); C07D
239/02 (20060101) |
Field of
Search: |
;544/335 ;514/256 |
Foreign Patent Documents
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1 110 957 |
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Jun 2001 |
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EP |
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WO 2006/120557 |
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Nov 2006 |
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WO |
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WO 2007/009524 |
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Jan 2007 |
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WO |
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1966, 5(5-6), 262-268. cited by other .
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Therapy", Clin. Cancer Res. 2006, 12, 6869-6875. cited by other
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Ellis, L.M. and D.J. Hicklin., "VEGF-targeted therapy: mechanisms
of anti-tumour activity", Nature Rev. Cancer 2008, 8, 579-591
[Published online Jul. 3, 2008]. cited by other .
Ferrara, N. and R.S. Kerbel, "Angiogenesis as a therapeutic
target", Nature 2005, 438, 967-974. cited by other .
Gaillard, P. et al., Design and Synthesis of the First Generation
of Novel Potent, Selective and in Vivo Active
(Benzothiazol-2-yl)acetonitrile Inhibitors of the c-Jun N-Terminal
Kinase, J. Med. Chem. 2005, 48, 4596-4607. cited by other .
Mortlock, A. et al., "Progress in the Development of Selective
Inhibitors of Aurora Kinases", Curr. Topics Med. Chem. 2005, 5,
199-213. cited by other .
Myrianthopoulos, V. et al., "An Integrated Computational Approach
to the Phenomenon of Potent and Selective Inhibition of Aurora
Kinases B and C by a Series of 7-Substituted Indirubins", J. Med.
Chem. 2007, 50(17), 4027-4037 [Published online Aug. 1, 2007].
cited by other .
Shaikh, A.R. et al., "Three-dimensional quantitative
structure-activity relationship (3 D-QSAR) and docking studies on
(benzothioazole-2-yl)acetonitrile derivatives as c-Jun N-terminal
kinase-3 (JNK-3) inhibitors", Bioorg. Med. Chem. Lett. 2006,
16(22), 5917-5925. cited by other.
|
Primary Examiner: Anderson; Rebecca
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit under 35 USC 119(e) of U.S.
Provisional Patent Application No. 61/100,869, filed 29 Sep. 2008,
entitled "2-[1H-benzimidazol-2(3H)-ylidene]- and
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides as
kinase inhibitors", the entire disclosure of which is incorporated
into this application by reference.
Claims
We claim:
1. A compound of the formula, ##STR00155## or a salt thereof,
where: X is NH or S; m is 0, 1, 2, or 3; n is 0, 1, 2, 3, or 4;
R.sup.1 is alkyl, substituted alkyl, heteroalkyl, substituted
heteroalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, aralkyl, substituted aralkyl, heteroaralkyl,
substituted heteroaralkyl, halo, nitro, or cyano, or is --OR, --SR,
--C(O)R, --OC(O)R, --C(O)OR, --NR.sub.2, --SO.sub.2OR,
--OSO.sub.2R, --SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R,
--CONR.sub.2, --NR.sup.3COR, or --NR.sup.3C(O)OR, where each R
independently is hydrogen, alkyl, substituted alkyl, heteroalkyl,
substituted heteroalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, aralkyl, substituted aralkyl,
heteroaralkyl, or substituted heteroaralkyl, and R.sup.3 is
hydrogen or C.sub.1-C.sub.3 alkyl; and R.sup.2 is alkyl,
substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, aralkyl,
substituted aralkyl, heteroaralkyl, substituted heteroaralkyl,
halo, nitro, or cyano, or is --OR, --SR, --C(O)R, --OC(O)R,
--C(O)OR, --NR.sub.2, --SO.sub.2OR, --OSO.sub.2R,
--SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R, --CONR.sub.2,
--NR.sup.3COR, or --NR.sup.3C(O)OR, where each R independently is
hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted
heteroalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, aralkyl, substituted aralkyl, heteroaralkyl, or
substituted heteroaralkyl, and R.sup.3 is hydrogen or
C.sub.1-C.sub.3 alkyl.
2. A compound of claim 1 where X is NH.
3. A compound of claim 1 where X is S.
4. A compound of claim 1 where m is at least 1.
5. A compound of claim 1 where m is 1.
6. A compound of claim 5 where R.sup.1 is on the 4-position of the
pyrimidine, and is methyl, methoxy, or trifluoromethyl.
7. A compound of claim 1 where m is 2.
8. A compound of claim 7 where one R.sup.1 is on the 4-position of
the pyrimidine, and is methyl, methoxy, or trifluoromethyl.
9. A compound of claim 8 where the other R.sup.1 is a group
selected from the --OR, --SR, --C(O)R, --OC(O)R, --C(O)OR,
--OSO.sub.2R, --SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R,
--CONR.sub.2, --NR.sup.3COR, and --NR.sup.3C(O)OR.
10. A compound of claim 1 where n is 0.
11. A compound of claim 1 where n is at least 1.
12. A compound of claim 1 where n is 1.
13. A compound of claim 12 where R.sup.2 is a group selected from
--OR, --SR, --C(O)R, --OC(O)R, --C(O)OR, --NR.sub.2, --SO.sub.2OR,
--OSO.sub.2R, --SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R,
--CONR.sub.2, --NR.sup.3COR, and --NR.sup.3C(O)OR.
14. A compound of claim 1 where n is 2.
15. A compound of claim 14 where one R.sup.2 is a group selected
from --OR, --SR, --C(O)R, --OC(O)R, --C(O)OR, --NR.sub.2,
--SO.sub.2OR, --OSO.sub.2R, --SO.sub.2NR.sub.2,
--NR.sup.3SO.sub.2R, --CONR.sub.2, --NR.sup.3COR, and
--NR.sup.3C(O)OR.
16. A compound of claim 1 selected from:
2-(5-{[3-(4-morpholinyl)propyl]aminocarbonyl}-1H-benzimidazol-2(3H)-ylide-
ne)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-(6-{[3-(4-morpholinyl)propyl]aminocarbonyl}-benzothiazol-2(3H)-ylidene)-
-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-(5-{[3-(4-morpholinyl)propyl](ethyl)aminocarbonyl}-1H-benzimidazol-2(3H-
)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-[5-(piperazine-1-carbonyl)-1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluor-
omethylpyrimidin-2-yl)acetamide,
2-{5-[(diethylaminomethyl)carbonylamino]-1H-benzimidazol-2(3H)-ylidene}-2-
-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(S)-(1-methylpyrrolidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-y-
lidene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(1-amino-1-methylethyl)carbonylamino]-1H-benzimidazol-2(3H)-ylidene-
}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(RS)-(1-methylpiperidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-y-
lidene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-({5-[2-(4-methylpiperazin-1-yl)ethyl]carbonylamino}-1H-benzimidazol-2(3-
H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-(6-{[2-(imidazol-1-yl)ethyl]benzothiazol-2(3H)-ylidene})-2-(4-trifluoro-
methylpyrimidin-2-yl)acetamide,
2-(6-{[2-(1,1-dioxothiomorpholin-4-yl)ethyl]benzothiazol-2(3H)-ylidene})--
2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-(6-{[2-(morpholin-4-yl)ethyl]benzothiazol-2(3H)-ylidene})-2-(4-trifluor-
omethylpyrimidin-2-yl)acetamide,
2-(6-{[2-(imidazol-1-yl)ethyloxy]benzothiazol-2(3H)-ylidene})-2-(4-triflu-
oromethylpyrimidin-2-yl)acetamide,
2-(1H-benzimidazol-2(3H)-ylidene)-2-[4-methyl-6-(trifluoromethyl)pyrimidi-
n-2-yl]acetamide,
2-(5-{[(diethylaminomethyl)carbonyl](ethyl)amino}-1H-benzimidazol-2(3H)-y-
lidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(S)-(1-methylpiperidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-yl-
idene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(R)-(1-methylpiperidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-yl-
idene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(S)-(1-methylpyrrolidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-y-
lidene}-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetamide,
2-(5-{[(S)-(1-methylpyrrolidin-2-yl)carbonyl](ethyl)amino}-1H-benzimidazo-
l-2(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, and
2-(5-{[(piperidin-1-ylmethyl)carbonyl](ethyl)amino}-1H-benzimidazol-2(3H)-
-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, or a salt
thereof.
17. A compound of claim 16 selected from:
2-(5-{[3-(4-morpholinyl)propyl](ethyl)aminocarbonyl}-1H-benzimidazol-2(3H-
)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(diethyl aminomethyl)carbonyl
amino]-1H-benzimidazol-2(3H)-ylidene}-2-(4-trifluoromethylpyrimidin-2-yl)-
acetamide,
2-({5-[2-(4-methylpiperazin-1-yl)ethyl]carbonylamino}-1H-benzim-
idazol-2(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-(5-{[(diethylaminomethyl)carbonyl](ethyl)amino}-1H-benzimidazol-2(3H)-y-
lidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(S)-(1-methylpiperidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-yl-
idene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide,
2-{5-[(R)-(1-methylpiperidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-yl-
idene}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, and
2-(5-{[(S)-(1-methylpyrrolidin-2-yl)carbonyl](ethyl)amino}-1H-benzimidazo-
l-2(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, or a
salt thereof.
18. A pharmaceutical composition comprising a therapeutically
effective amount of a compound of claim 1, in combination with an
excipient.
19. A method of treatment of colon cancer comprising administration
of a compound of claim 1 to a subject in need thereof.
20. A method of treatment of promyelocytic leukemia comprising
administration of a compound of claim 1 to a subject in need
thereof.
21. A method for inhibiting an aurora kinase or a VEGFR2 kinase
which method comprises contacting the kinase with an inhibitory
amount of a compound of claim 1.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides and
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides, their
salts, pharmaceutical compositions containing them, and their use
as kinase inhibitors and in the treatment of cancer.
2. Description of the Related Art
As discussed by Mortlock et al., "Progress in the Development of
Selective Inhibitors of Aurora Kinases", Curr. Topics Med. Chem.
2005, 5, 199-213, and Carvajal et al., "Aurora Kinases: New Targets
for Cancer Therapy", Clin. Cancer Res. 2006, 12, 6869-6875, the
Aurora family of kinases are involved in the regulation of mitosis.
Two of the three human Aurora kinases, Aurora A and Aurora B, are
frequently overexpressed in human tumors, while the Aurora A gene
itself is amplified in many tumors. There has thus been
considerable interest in the development of inhibitors of Aurora
kinases as anticancer compounds for the treatment of both solid
malignancies (e.g., colorectal, lung, breast, pancreatic, and
bladder cancer) and hematological malignancies (e.g., acute
lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML),
Hodgkin's and non-Hodgkin's lymphomas, and myelodysplastic syndrome
(MDS)). A number of compounds have reached clinical trials,
including Vertex and Merck's VX-680/MK-0457, which reached Phase II
clinical studies but has now been discontinued and replaced in
development by a later compound, VX-689 (MK-5108); Astex's AT9283;
Astra Zeneca's AZD-1152; Entremed's ENMD-2076 and ENMD-981693;
Millennium's MLN-8054 and MLN-8237; Nerviano's PHA-739358; Rigel's
R763; Sunesis's SNS-314; and others.
As discussed by Ferrera and Kerbel, "Angiogenesis as a therapeutic
target", Nature 2005, 438, 967-974, and more recently by Ellis and
Hicklin, "VEGF-targeted therapy: mechanisms of anti-tumour
activity", Nature Rev. Cancer 2008, 8, 579-591, the inhibition of
angiogenesis by the targeting of VEGF-A and its receptors is
considered to be a highly promising strategy for cancer treatment.
Two inhibitors of VEGFR2 kinase; sunitinib (Pfizer's Sutent.RTM.)
and sorafenib (Bayer/Onyx's Nexavar.RTM.) are already approved in
the US for the treatment of kidney cancer, while sunitinib is also
approved for the treatment of gastro-intestinal cancer. A number of
other VEGFR2 kinase-inhibiting compounds are in advanced
development, among them GSK's pazopanib, Novartis's vatalinib,
Pfizer's axitinib, Astra Zeneca's vandetanib, and others, for both
solid (e.g. breast, ovarian, lung, and colorectal) and
hematological malignancies. Also, bevacizumab (Genentech's
Avastin), although not a VEGFR2 kinase inhibitor but an anti-VEGF
antibody, is seeing use in brain cancer, suggesting that VEGFR2
kinase inhibitors may also find use in it, as in all other solid
tumors.
Many of these compounds, whether the Aurora kinase inhibitors or
the VEGFR inhibitors mentioned in the above paragraphs, are not
specific inhibitors of the named kinases, but rather are selective
inhibitors of the named kinases, also acting on other kinases. For
example, sorafenib and sunitinib also have significant activity
against Raf kinase and other kinases, and Cyclacel's CYC116 has
been reported to inhibit Aurora kinases A, B, and C, VEGFR2 kinase,
and Flt3 with IC.sub.50s all below 100 nM.
It would be desirable to develop compounds that are potent
inhibitors of Aurora kinase, also inhibiting VEGFR2 kinase, as
anticancer agents.
The disclosures of the documents referred to in this application
are incorporated into this application by reference.
SUMMARY OF THE INVENTION
In a first aspect, this invention is compounds of formula A:
##STR00001## and their salts, where: X is NH or S; m is 0, 1, 2, or
3; n is 0, 1, 2, 3, or 4; R.sup.1 is alkyl, substituted alkyl,
heteroalkyl, substituted heteroalkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, aralkyl, substituted aralkyl,
heteroaralkyl, substituted heteroaralkyl, halo, nitro, or cyano, or
is --OR, --SR, --C(O)R, --OC(O)R, --C(O)OR, --NR.sub.2,
--SO.sub.2OR, --OSO.sub.2R, --SO.sub.2NR.sub.2,
--NR.sup.3SO.sub.2R, --CONR.sub.2, --NR.sup.3COR, and
--NR.sup.3C(O)OR, where each R independently is hydrogen, alkyl,
substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, aralkyl,
substituted aralkyl, heteroaralkyl, or substituted heteroaralkyl,
and R.sup.3 is hydrogen or C.sub.1-C.sub.3 alkyl; and R.sup.2 is
alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
aralkyl, substituted aralkyl, heteroaralkyl, substituted
heteroaralkyl, halo, nitro, or cyano, or is --OR, --SR, --C(O)R,
--OC(O)R, --C(O)OR, --NR.sub.2, --SO.sub.2OR, --OSO.sub.2R,
--SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R, --CONR.sub.2,
--NR.sup.3COR, and --NR.sup.3C(O)OR, where each R independently is
hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted
heteroalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, aralkyl, substituted aralkyl, heteroaralkyl, or
substituted heteroaralkyl, and R.sup.3 is hydrogen or
C.sub.1-C.sub.3 alkyl.
In second through sixth aspects, this invention is compounds of the
first aspect of this invention for use as kinase inhibitors,
especially as inhibitors of Aurora kinase (notably Aurora kinases A
and B) and optionally VEGFR2 kinase, pharmaceutical compositions
containing the compounds of the first aspect of this invention, use
of the compounds of the first aspect of this invention as kinase
inhibitors and for the manufacture of medicaments, and methods of
treatment using the compounds of the first aspect of this
invention.
In a seventh aspect, this invention is methods of making the
compounds of the first aspect of this invention.
Preferred embodiments of this invention are characterized by the
specification including the examples.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
"Alkyl" means a monovalent group derived from a saturated or
unsaturated (but not aromatically unsaturated) C.sub.1-C.sub.10
hydrocarbon that may be linear, branched, or cyclic, by removal of
one hydrogen atom from a carbon atom. Examples are methyl, ethyl,
propyl, 1-propenyl, isopropyl, butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, hexyl, cyclopentyl, cyclopenten-1-yl,
cyclopropylmethyl, cyclohexyl, and cyclohexylmethyl. Saturated
alkyls (including cycloalkyls) and C.sub.1-C.sub.6 alkyls ("lower
alkyls"), especially C.sub.1-C.sub.3 alkyls, are exemplary. Note
that the definition of "alkyl" in this application is broader than
the conventional definition and includes groups more commonly
referred to as "cycloalkyl", "cycloalkylalkyl", "alkenyl", and
"alkynyl".
A "substituted alkyl" is an alkyl substituted with up to three
halogen atoms and/or up to three substituents selected from nitro,
cyano, --OR, --SR, --COR, --OC(O)R, --C(O)OR, --NR.sub.2,
--SO.sub.2OR, --OSO.sub.2R, --SO.sub.2NR.sub.2, --NRSO.sub.2R,
--CONR.sub.2, --NRCOR, and --NRC(O)OR, where each R independently
is hydrogen, optionally R'-substituted alkyl, optionally
R'-substituted heteroalkyl, optionally R'-substituted aryl,
optionally R'-substituted heteroaryl, optionally R'-substituted
aralkyl, or optionally R'-substituted heteroaralkyl and each R'
independently is 1 to 3 substituents selected from halo, nitro,
cyano, hydroxy, mercapto, amino, cycloamino, C.sub.1-3 alkyl,
C.sub.1-3 alkyloxy, or --C(O)O--C.sub.1-3 alkyl (preferably, 1 to 3
substituents selected from halo, nitro, cyano, hydroxy, mercapto,
amino, cycloamino, C.sub.1-3 alkyl, or C.sub.1-3 alkyloxy), or two
R groups together form a 4- to 6-member optionally R'-substituted
alkanediyl or optionally R'-substituted heteroalkanediyl
(especially where the nitrogen and the two R groups of --NR.sub.2
form a cycloamino group). Thus, for example, substituted alkyl
groups include such groups as trifluoromethyl, 3-chloropropyl, and
2-(morpholin-4-yl)ethyl.
"Heteroalkyl" means alkyl in which 1 to 3 of the carbon atoms are
replaced by O, S, SO.sub.2, or NR (where R is H or C.sub.1-3 alkyl
optionally substituted with halogen or hydroxy), including linear
groups such as 3-oxapentyl; monocyclic rings containing 5 or 6 ring
atoms such as 2-tetrahydrofuranyl, 2-pyrrolidinyl, 3-piperidinyl,
2-piperazinyl, 4-methyl-1-piperazinyl, 4-dihydropyranyl, and
3-morpholinyl; and groups such as tetrahydrofuran-2-ylmethyl and
piperidin-3-ylethyl. Heteroalkyl groups also include those where a
ring nitrogen is oxidized to form an N-oxide. "Substituted
heteroalkyl" means heteroalkyl substituted in the manner described
above for substituted alkyl. A "cycloamino" group is a cyclic
heteroalkyl of 5 to 7 ring atoms containing a nitrogen ring atom by
which the group is bonded to the remainder of the molecule of which
it forms a part and optionally containing a further ring heteroatom
selected from O, S, SO.sub.2, and NR (where R is H or C.sub.1-3
alkyl optionally substituted with halogen, hydroxy, or 1 or 2
phenyl groups). 4-Methyl-1-piperazinyl,
4-(2-hydroxyethyl)-1-piperazinyl, 4-(diphenylmethyl)-1-piperazinyl,
and 4-morpholinyl are examples of cycloamino groups. Compounds of
this invention also include compounds where any --NR.sub.2 group
present is replaced by a cycloamino group.
"Aryl" means a monovalent group derived from an aromatic
hydrocarbon containing 6 to 14 ring carbon atoms by removal of one
hydrogen atom from a carbon atom, which is monocyclic (e.g.,
phenyl), condensed polycyclic, for example, condensed bicyclic
(e.g., naphthyl), or linked polycyclic, for example, linked
bicyclic (e.g., biphenylyl). A preferred aryl is phenyl.
"Substituted aryl" means aryl substituted with up to three
substituents selected from halo, nitro, cyano, --OR, C.sub.1-3
alkyl or C.sub.1-3 alkyloxy (each optionally substituted with halo
or --NR.sub.2), --SR, --COR, --OC(O)R, --C(O)OR, --NR.sub.2,
--SO.sub.2OR, --OSO.sub.2R, --SO.sub.2NR.sub.2, --NRSO.sub.2R,
--CONR.sub.2, --NRCOR, and --NRC(O)OR, where each R independently
is hydrogen, optionally R'-substituted alkyl, optionally
R'-substituted heteroalkyl, optionally R'-substituted aryl,
optionally R'-substituted heteroaryl, optionally R'-substituted
aralkyl, or optionally R'-substituted heteroaralkyl (preferably,
hydrogen or optionally R'-substituted alkyl) and each R'
independently is 1 to 3 substituents selected from halo, nitro,
cyano, hydroxy, mercapto, amino, cycloamino, C.sub.1-3 alkyl,
C.sub.1-3 alkyloxy, or --C(O)Oalkyl (preferably, halo, nitro,
cyano, hydroxy, mercapto, amino, cycloamino, C.sub.1-3 alkyl, or
C.sub.1-3 alkyloxy), or two R groups together form a 4- to 6-member
optionally R'-substituted alkanediyl or optionally R'-substituted
heteroalkanediyl (especially where the nitrogen and the two R
groups of --NR.sub.2 form a cycloamino group). Two adjacent
substituents may also form a methylenedioxy or ethylenedioxy group.
Substituted aryl groups include aryl groups substituted with up to
three substituents selected from the group consisting of halo,
nitro, cyano, hydroxy, mercapto, amino, optionally halo-substituted
C.sub.1-3 alkyl, and optionally halo-substituted C.sub.1-3
alkyloxy, for example, phenyl substituted in this way. Preferred
substituted aryls are substituted phenyls.
"Aralkyl" means alkyl substituted with aryl, such as benzyl and
phenethyl. A preferred aralkyl is benzyl. "Substituted aralkyl"
means aralkyl in which one or both of the aryl and the alkyl are
substituted in the manner described above for substituted aryl and
substituted alkyl. Preferred substituted aralkyls are substituted
benzyls.
"Halogen" or "halo" means F, Cl, Br, I; particularly F or Cl.
"Heteroaryl" means an aromatic monovalent group derived from a
cyclic hydrocarbon containing 5 to 14 ring atoms in which 1 to 4
(preferably 1 to 3) of the ring carbon atoms are replaced by O, S,
N, or NR (where R is H or C.sub.1-3 alkyl), preferably O, S, or NR,
by removal of one hydrogen atom from a ring carbon atom; including
monocyclic groups containing 5 or 6 ring atoms such as furanyl,
thienyl, pyrrolyl, oxazolyl, imidazolyl, pyridinyl, pyrazinyl,
pyridazinyl, pyrimidinyl, and the like, and bicyclic groups such as
benzothiazolyl, purinyl, and benzimidazolyl. Monocyclic rings are
preferred. Heteroaryl groups also include those where a ring
nitrogen is oxidized to form an N-oxide. "Substituted heteroaryl"
means heteroaryl substituted in the manner described above for
substituted aryl.
"Heteroaralkyl" means alkyl substituted with heteroaryl, such as
2-thienylmethyl. "Substituted heteroaralkyl" means heteroaralkyl
substituted in the manner described above for substituted
aralkyl.
"Elaborated" refers to the conversion of a reactive substituent to
another typically more complex substituent, such as the conversion
of an amine to an amide or sulfonamide, a carboxy group to an ester
or amide, a hydroxy to an ester, and conversion of an amide or
sulfonamide with one or more hydrogen atoms on the nitrogen to one
where one or more of those hydrogen atoms is replaced by an
optionally substituted alkyl, heteroalkyl, aryl, heteroaryl,
aralkyl, or heteroaralkyl group. "Protected" has its conventional
meaning in organic synthesis, namely the temporary conversion of a
reactive substituent to a substituent that is non-reactive under
the conditions of the reaction(s) proposed to be carried out; such
as the protection of an amine as a carbamate.
A "solubility-enhancing group" is a group that enhances the
solubility in water of the compound over a compound that is not so
substituted, for example, hydroxy and salt-forming groups such as
carboxy and non-amide amino groups, especially non-amide amino
groups that are capable of forming acid addition salts; or is a
group containing one or more of these groups as functional
subgroups, such as lower alkyl substituted with hydroxy. Preferred
solubility-enhancing groups include --NR'.sub.2 (where each R'
independently is hydrogen or C.sub.1-C.sub.3 alkyl, or where
--NR'.sub.2 together is cycloamino) or a nitrogen-containing
heteroaryl; lower alkyl substituted with --NR'.sub.2 (where each R'
independently is hydrogen or C.sub.1-C.sub.3 alkyl, or where
--NR'.sub.2 together is cycloamino) or with a nitrogen-containing
heteroaryl; and groups of the formula --OR, --SR, --C(O)R,
--OC(O)R, --C(O)OR, --NR.sub.2, --SO.sub.2OR, --OSO.sub.2R,
--SO.sub.2NR.sub.2, --NR.sup.3SO.sub.2R, --CONR.sub.2,
--NR.sup.3COR, and --NR.sup.3C(O)OR, where R is lower alkyl
substituted with --NR'.sub.2 (where each R' independently is H or
C.sub.1-C.sub.3 alkyl, or where --NR'.sub.2 together is cycloamino)
or with a nitrogen-containing heteroaryl, and R.sup.3 is hydrogen
or C.sub.1-C.sub.3 alkyl.
"Salts" are described in the section entitled "Compounds of this
invention".
A "therapeutically effective amount" means that amount which, when
administered to a human for treating a cancer, is sufficient to
effect treatment for the cancer. "Treating" or "treatment" of a
cancer in a human includes one or more of:
(1) limiting/inhibiting growth of the cancer, i.e.,
limiting/arresting its development,
(2) reducing/preventing spread of the cancer, i.e.
reducing/preventing metastases,
(3) relieving the cancer, i.e., causing regression of the
cancer,
(4) reducing/preventing recurrence of the cancer, and
(5) palliating symptoms of the cancer.
"Combination therapy" means the administration of a compound of the
first aspect of this invention and another anticancer therapy
during the course of cancer chemotherapy. Such combination therapy
may involve the administration of the compound of the first aspect
of this invention before, during, and/or after the administration
of the another anticancer therapy. The administration of the
compound of the first aspect of this invention may be separated in
time from the administration of the another anticancer therapy by
up to several weeks, and may precede it or follow it, but more
commonly the administration of the compound of the first aspect of
this invention will accompany at least one aspect of the another
anticancer therapy (such as the administration of one dose of a
chemotherapeutic agent, molecular targeted therapy agent, biologic
therapy agent, or radiation therapy) within up to 48 hours, and
most commonly within less than 24 hours.
"Comprising" or "containing" and their grammatical variants are
words of inclusion and not of limitation and mean to specify the
presence of stated components, groups, steps, and the like but not
to exclude the presence or addition of other components, groups,
steps, and the like. Thus "comprising" does not mean "consisting
of", "consisting substantially of", or "consisting only of"; and,
for example, a formulation "comprising" a compound must contain
that compound but also may contain other active ingredients and/or
excipients.
Compounds of this Invention
Salts (for example, pharmaceutically acceptable salts) of the
compounds of formula A are included in the present invention and
are useful in the compositions, methods, and uses described in this
application. Such salts are preferably formed with pharmaceutically
acceptable acids. See, for example, Stahl and Wermuth, eds.,
"Handbook of Pharmaceutically Acceptable Salts", (2002), Verlag
Helvetica Chimica Acta, Zurich, Switzerland, for an extensive
discussion of pharmaceutical salts, their selection, preparation,
and use. Unless the context requires otherwise, reference to any
compound of this invention is a reference both to the compound and
to its salts.
These salts include salts that may be formed when acidic protons
present are capable of reacting with inorganic or organic bases.
Typically the parent compound is treated with an excess of an
alkaline reagent, such as hydroxide, carbonate or alkoxide,
containing an appropriate cation. Cations such as Na.sup.+,
K.sup.+, Ca.sup.2+, Mg.sup.2+ and NH.sub.4.sup.+ are examples of
cations present in pharmaceutically acceptable salts. Suitable
inorganic bases, therefore, include calcium hydroxide, potassium
hydroxide, sodium carbonate and sodium hydroxide. Salts may also be
prepared using organic bases, such as salts of primary, secondary
and tertiary amines, substituted amines including
naturally-occurring substituted amines, and cyclic amines including
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine,
lysine, arginine, histidine, caffeine, procaine, hydrabamine,
choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines,
theobromine, purines, piperazine, piperidine, N-ethylpiperidine,
and the like.
If a compound of the first aspect of this invention contains a
basic group, such as an amino group, it may be prepared as an acid
addition salt. Acid addition salts of the compounds are prepared in
a standard manner in a suitable solvent from the parent compound
and an excess of an acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid (giving the sulfate and bisulfate salts),
nitric acid, phosphoric acid and the like, and organic acids such
as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,
mandelic acid, methanesulfonic acid, ethanesulfonic acid, salicylic
acid, 4-toluenesulfonic acid, hexanoic acid, heptanoic acid,
cyclopentanepropionic acid, lactic acid,
2-(4-hydroxybenzoyl)benzoic acid, 1,2-ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
camphorsulfonic acid, 4-methylbicyclo[2.2.2.]oct-2-ene-1-carboxylic
acid, glucoheptonic acid, gluconic acid,
4,4'-methylenebis(3-hydroxy-2-naphthoic)acid, 3-phenylpropionic
acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric
acid, glucuronic acid, glutamic acid, 3-hydroxy-2-naphthoic acid,
stearic acid, muconic acid and the like.
Compounds of this invention include those compounds of formula A
where one or more of the following is true:
1.a X is S; or
1.b X is NH;
2. m is at least 1, preferably 1 or 2;
3. the R.sup.1 group (when m is 1) or an R.sup.1 group is a group
on the 4-position (i.e. adjacent to one of the two nitrogen atoms
on the pyrimidine ring: may be referred to as the 6-position if m
is 2 or 3 and another substituent has naming priority), such as
4-methyl, 4-methoxy, or 4-trifluoromethyl (preferred); 4. when m is
2, the other R.sup.1 group is at the 5- or 6-position, especially
the 6-position (defined when the first R.sup.1 group is at the
4-position); typically a group that is either small (e.g. methyl,
methoxy, carboxyl, or C.sub.1-C.sub.3 alkoxycarbonyl), or a group
that enhances a physicochemical property of the molecule, such as a
solubility-enhancing group; 5. n is at least 1, preferably 1 or 2;
6. the R.sup.2 group (when n is 1) or an R.sup.2 group (when n is
at least 2) is a group that enhances a physicochemical property of
the molecule, such as a solubility-enhancing group.
Generally, a compound having a greater number of these features is
preferred over a compound having a lesser number of these features;
in particular, addition of one of these features to a compound
having less than all the features will generally result in a
compound that is preferred over the compound without that
feature.
Tautomerism and Naming
The compounds of this invention are named and shown in this
application as
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides and
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamides, i.e.
compounds having an exocyclic double bond between the
benzimidazole/benzothiazole section and the
2-(pyrimidin-2-yl)acetamide section of the molecule. However, as
will be obvious to a person having ordinary skill in the art, the
compounds are tautomeric and may also be named and shown as having
the double bond within the imidazole/thiazole ring and the
exocyclic bond to the 2-(pyrimidin-2-yl)acetamide being single.
Also, because there is no inherent chiral preference at the
2-carbon of the acetamide when the exocyclic bond becomes single,
and because the 2-(pyrimidin-2-yl)acetamide section of the molecule
may rotate relative to the benzimidazole/benzothiazole section when
the exocyclic bond is single, the E and Z isomers when the
exocyclic bond is double also become tautomeric. Additional
keto-enol tautomerism at the amide, and within the pyrimidine ring,
is also possible. Further, there is additional symmetry within the
benzimidazole such that the 4- and 7-positions, and the 5- and
6-positions, on the benzimidazole are equivalent. Thus the
compounds may adopt a wide variety of at least potentially
interchangeable conformations, and the illustration or naming of a
compound in this specification and claims in a particular
conformation is not intended to be limited to that conformation but
is intended to include all conformations applicable to that
compound.
For parallelism, each of the compounds of formula A is named in
this specification and claims as a derivative of
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide rather
than by following the priority rules of IUPAC naming conventions.
Thus, for example, compound 39A, the compound of the formula
##STR00002## is named
2-(5-{[3-(4-morpholinyl)propyl]aminocarbonyl}-1H-benzimidazol-2
(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, and
compound 40A, the compound of the formula
##STR00003## is named
2-(6-{[3-(4-morpholinyl)propyl]aminocarbonyl}-benzothiazol-2(3H)-ylidene)-
-2-(4-trifluoromethylpyrimidin-2-yl)acetamide.
Compounds of this invention include each of the compounds described
in the specification and claims of this application as filed,
including in the Examples and the compound table below, such as any
one of compounds 1A to 146A, especially any one of compounds 39A to
41A, 60A, 75A, 77A, 81A, 82A, 83A, 95A, 100A, 101A, 117A, 138A,
139A, and 142A to 146A, and their salts; particularly any one of
compounds 41A, 75A, 83A, 139A, 142A, 143A, and 145A, and their
salts. Compositions and methods, etc., of this invention include
compositions and methods, etc., where the compound is one of those
mentioned in the preceding sentence.
Preparation of the Compounds
In each of the Reaction Schemes shown below in the discussion of
the general synthetic methods, no substituents are shown on the
benzene ring of either the benzimidazole/benzothiazole or on the
pyrimidine, but it will be apparent that substituents (either the
final substituents on the desired compound, or precursors to those
final substituents to be modified after formation of the compound
core) may be present, as discussed later in the specification and
as illustrated by the examples.
A first general synthetic method, applicable to both the
benzothiazole- and benzimidazole-based compounds, involves the
formation of 2-[1H-benzimidazol-2(3H)-ylidene]acetonitrile or
2-[benzothiazol-2(3H)-ylidene]acetonitrile, followed by coupling
with a pyrimidine and hydrolysis of the nitrile to the amide (in
either order), and is illustrated in Reaction Scheme 1 below.
##STR00004##
In the first part of the synthesis, in the first step, a
benzene-1,2-diamine (1, X.dbd.NH) or 2-aminothiophenol (1, X.dbd.S)
is coupled with a
2-(alkoxycarbonyl)-3,3-bis(methylthio)acrylonitrile 2, such as the
ethyl or tert-butyl ester, to give the
2-cyano-2-[1H-benzimidazol-2(3H)-ylidene]acetate or
2-cyano-2-[benzothiazol-2(3H)-ylidene]acetate 3. The reaction is
carried out in an alkanol, e.g. ethanol, optionally in the presence
of an amine base, such as 4-dimethylaminopyridine, under heating.
In the second step, the alkoxycarbonyl group is removed by
hydrolysis, for example basic hydrolysis to remove an
ethoxycarbonyl group or acidic hydrolysis to remove the preferred
tert-butoxycarbonyl group, giving the
2-[1H-benzimidazol-2-yl]acetonitrile or
2-[benzothiazol-2-yl]acetonitrile 4.
2-(Ethoxycarbonyl)-3,3-bis(methylthio)acrylonitrile is commercially
available and its preparation described in the literature; the
tert-butyl analog may readily be prepared by the same method.
In the second part of the synthesis, the
2-[1H-benzimidazol-2-yl]acetonitrile or
2-[benzothiazol-2-yl]acetonitrile 4 is either coupled with a
pyrimidine and then hydrolyzed to the amide, or hydrolyzed and then
coupled. If compound 4 is a 2-[1H-benzimidazol-2-yl]acetonitrile,
one of the benzimidazole nitrogen atoms may be protected with an
amine-protecting group, such as with tert-butoxycarbonyl,
preventing N-alkylation. In the coupling/hydrolysis route, compound
4 is then coupled with a pyrimidine 5 [L is a leaving group such as
Cl, methylsulfonyl, or methylthio] in the presence of a strong
base, such as sodium hydride or lithium hexamethyldisilazane, in an
aprotic polar solvent, such as tetrahydrofuran, to give a
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)-acetonitrile
or 2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile 6.
Compound 6 is then hydrolyzed with concentrated sulfuric acid to
give the final
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide 8. In
the hydrolysis/coupling route, the steps of the preceding sentence
are reversed: the nitrile of compound 4 is hydrolyzed to the amide,
giving compound 7, and compound 7 is then coupled with the
pyrimidine 5 to give compound 8.
A second general synthetic method, also applicable to both the
benzothiazole- and benzimidazole-based compounds, involves the
formation of a
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile,
followed by hydrolysis of the nitrile to the amide, and is
illustrated in Reaction Scheme 2 below.
##STR00005##
In the first step of the synthesis, a benzene-1,2-diamine (1,
X.dbd.NH) or 2-aminothiophenol (1, X.dbd.S) is coupled with a
3,3-bis(methylthio)-2-(pyrimidin-2-yl)acrylonitrile 9 to give a
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile 6.
The reaction is carried out under generally the same conditions as
are used in the first step of the first half of Reaction Scheme 1.
In the second step, the compound of formula 6 is hydrolyzed to a
2-[1H-benzimidazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide 8, as in
Reaction Scheme 1.
As shown in Reaction Scheme 2, the
3,3-bis(methylthio)-2-(pyrimidin-2-yl)acrylonitrile 9 may be
prepared by the coupling of a pyrimidine with a 2-cyanoacetate
ester (for example the tert-butyl ester), followed by removal of
the ester group to give the 2-(2-pyrimidinyl)acetonitrile 11, and
reaction of that with a xanthate salt followed by methyl iodide to
give the dithioketal. The preparation of
3,3-bis(methylthio)-2-(4-trifluoromethylpyrimidin-2-yl)acrylonitrile
is given in Preparative Example 4.
A third general synthetic method, applicable to the
benzothiazole-based compounds, involves the coupling of a
2-(pyrimidin-2-yl)acetonitrile or 2-(pyrimidin-2-yl)acetamide with
a 2-chloro-benzothiazole, followed by hydrolysis of the nitrile if
necessary, as shown in Reaction Scheme 3.
##STR00006##
In this synthesis, the benzothiazole 10 [L is a leaving group] is
coupled with a 2-(pyrimidin-2-yl)acetonitrile 11 or
2-(pyrimidin-2-yl)acetamide 12 in the presence of a strong base,
such as sodium hydride, in an aprotic polar solvent, such as
tetrahydrofuran, to give a
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile 6 or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide 8. If
the compound of formula 6 has been prepared, it is then hydrolyzed
to a compound of formula 8, as in Reaction Scheme 1.
A fourth general synthetic method, also applicable to the
benzothiazole-based compounds, also involves coupling of a
2-(pyrimidin-2-yl)acetonitrile or 2-(pyrimidin-2-yl)acetamide, here
with a phenyl isothiocyanate, typically formed from an aniline,
followed by ring closure and hydrolysis of the nitrile if
necessary, as shown in Reaction Scheme 4. This method is
particularly attractive for compounds with complex sidechains on
the benzene ring of the benzothiazole because the preparation of
substituted anilines is well known.
##STR00007##
In the first step of the synthesis, the phenyl isothiocyanate 14
(which may be readily prepared by reaction of an aniline with
thiophosgene) is coupled with a 2-(pyrimidin-2-yl)acetonitrile 11
or 2-(pyrimidin-2-yl)acetamide 12 in the presence of a base to give
a
3-(phenylamino)-2-(pyrimidin-2(1H)-ylidene)-3-thioxo-propionitrile
15 or 3-(phenylamino)-2-(pyrimidin-2-yl)-3-thioxopropanamide 16. In
the second step, ring closure of the compound of formula 15 or 16
is achieved by reaction with an agent such as bromine/acetic acid
or potassium ferricyanide, to give a
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile 6 or
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide 8. If
the compound of formula 6 has been prepared, it is then hydrolyzed
to a compound of formula 8, as in Reaction Scheme 1.
A fifth general synthetic method, also applicable to the
benzothiazole-based compounds, involves the formation of a
2-[benzothiazol-2(3H)-ylidene]malononitrile, followed by conversion
of one of the nitrile groups to an imidate and then to an amidine.
The amidine is reacted with a .beta.-diketone or equivalent to form
the pyrimidine ring, and the other nitrile hydrolyzed to the
corresponding carboxamide; or the amidine-nitrile may be hydrolyzed
and then reacted with the .beta.-diketone or equivalent, as shown
in Reaction Scheme 5. This method is particularly attractive for
forming compounds where the substituted pyrimidine is not available
as a starting material.
##STR00008##
In the first step, the benzothiazole 10 [L is a leaving group, as
in Reaction Scheme 4] reacts with malononitrile in the presence of
a strong base, such as sodium ethoxide in ethanol, to give the
2-[benzothiazol-2(3H)-ylidene]malononitrile 17. Reaction of this
nitrile in suspension in a polar solvent with gaseous hydrogen
chloride gives the
2-[benzothiazol-2(3H)-ylidene]-2-cyanoacetimidate 18, and reaction
of the imidate with ammonia gives the corresponding
2-[benzothiazol-2(3H)-ylidene]-2-cyanoacetamidine 19. Compound 19
is then reacted with a .beta.-diketone or equivalent, such as
2,4-pentanedione or ethyl 3-oxo-4,4,4-trifluorobutyrate, to form
the pyrimidine ring [substituents not shown] of the compound of
formula 6, in the presence of a base such as sodium ethoxide in an
aprotic solvent under strong (e.g. microwave) heating. Finally, the
compound of formula 6 is hydrolyzed to a compound of formula 8, as
in Reaction Scheme 1. If the order is reversed, so that the
hydrolysis is performed before the coupling, the hydrolysis
proceeds as in Reaction Scheme 1, and the coupling may take place
under milder conditions.
Compounds of formula A may be converted to salts by reaction with
the appropriate acids, using techniques well known to a person of
ordinary skill in the art for the formation of acid addition salts.
The acid used, and the reaction conditions, may be chosen to give
salts that are pharmaceutically acceptable and that have a form
convenient for isolation and formulation, such as a solid form (for
example, amorphous or crystalline).
Compounds for a Use, Compositions, and Uses
The second aspect of this invention is the compounds of the first
aspect of this invention for use as kinase inhibitors, especially
as an inhibitor of Aurora kinase and optionally VEGFR2 kinase,
particularly for the treatment of cancer. The third aspect of this
invention is pharmaceutical compositions comprising a compound of
the first aspect of this invention and optionally a
pharmaceutically acceptable excipient. The fourth aspect of this
invention is the use of the compounds of the first aspect of this
invention as kinase inhibitors; and the fifth aspect of this
invention is the use of the compounds in the manufacture of
medicaments for kinase inhibition, especially for the treatment of
cancer.
The compounds of the first aspect of this invention may be
administered by any route suitable to the subject being treated and
the nature of the subject's condition. Routes of administration
include administration by injection, including intravenous,
intraperitoneal, intramuscular, and subcutaneous injection, by
transmucosal or transdermal delivery, through topical applications,
nasal spray, suppository and the like or may be administered
orally. Formulations may optionally be liposomal formulations,
emulsions, formulations designed to administer the drug across
mucosal membranes or transdermal formulations. Suitable
formulations for each of these methods of administration may be
found, for example, in Remington: The Science and Practice of
Pharmacy, 20th ed., A. Gennaro, ed., Lippincott Williams &
Wilkins, Philadelphia, Pa., U.S.A. Typical formulations will be
either oral or solutions for intravenous infusion. Typical dosage
forms will be tablets or capsules for oral administration,
solutions for intravenous infusion, and lyophilized powders for
reconstitution as solutions for intravenous infusion.
Depending on the intended mode of administration, the
pharmaceutical compositions may be in the form of solid, semi-solid
or liquid dosage forms, preferably in unit dosage form suitable for
single administration of a precise dosage. In addition to an
effective amount of the active compound(s), the compositions may
contain suitable pharmaceutically-acceptable excipients, including
adjuvants which facilitate processing of the active compounds into
preparations which can be used pharmaceutically. "Pharmaceutically
acceptable excipient" refers to an excipient or mixture of
excipients which does not interfere with the effectiveness of the
biological activity of the active compound(s) and which is not
toxic or otherwise undesirable to the subject to which it is
administered.
For solid compositions, conventional excipients include, for
example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharin, talc, cellulose, glucose,
sucrose, magnesium carbonate, and the like. Liquid
pharmacologically administrable compositions can, for example, be
prepared by dissolving, dispersing, etc., an active compound as
described herein and optional pharmaceutical adjuvants in water or
an aqueous excipient, such as, for example, water, saline, aqueous
dextrose, and the like, to form a solution or suspension. If
desired, the pharmaceutical composition to be administered may also
contain minor amounts of nontoxic auxiliary excipients such as
wetting or emulsifying agents, pH buffering agents and the like,
for example, sodium acetate, sorbitan monolaurate, triethanolamine
sodium acetate, triethanolamine oleate, etc.
For oral administration, the composition will generally take the
form of a tablet or capsule, or it may be an aqueous or nonaqueous
solution, suspension or syrup. Tablets and capsules are preferred
oral administration forms. Tablets and capsules for oral use will
generally include one or more commonly used excipients such as
lactose and corn starch. Lubricating agents, such as magnesium
stearate, are also typically added. When liquid suspensions are
used, the active agent may be combined with emulsifying and
suspending excipients. If desired, flavoring, coloring and/or
sweetening agents may be added as well. Other optional excipients
for incorporation into an oral formulation include preservatives,
suspending agents, thickening agents, and the like.
Injectable formulations can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solubilization or suspension in liquid prior to injection, or as
emulsions or liposomal formulations. The sterile injectable
formulation may also be a sterile injectable solution or a
suspension in a nontoxic parenterally acceptable diluent or
solvent. Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution and isotonic sodium chloride
solution. In addition, sterile, fixed oils, fatty esters or polyols
are conventionally employed as solvents or suspending media.
The pharmaceutical compositions of this invention may also be
formulated in lyophilized form for parenteral administration.
Lyophilized formulations may be reconstituted by addition of water
or other aqueous medium and then further diluted with a suitable
diluent prior to use. The liquid formulation is generally a
buffered, isotonic, aqueous solution. Examples of suitable diluents
are isotonic saline solution, 5% dextrose in water, and buffered
sodium or ammonium acetate solution. Pharmaceutically acceptable
solid or liquid excipients may be added to enhance or stabilize the
composition, or to facilitate preparation of the composition.
Typically, a pharmaceutical composition of the present invention is
packaged in a container with a label, or instructions, or both,
indicating use of the pharmaceutical composition in the treatment
of cancer.
The pharmaceutical composition may additionally contain one or more
other pharmacologically active agents in addition to a compound of
this invention. These additional active agents will typically be
useful in treating cancer, or for enhancing the treatment of cancer
by compounds of this invention.
Methods of Using the Compounds
The compounds of the first aspect of this invention have activity
against human cancer cell lines, as demonstrated in the in vitro
and in vivo Examples below, and are therefore considered to be
useful as human cancer chemotherapeutic agents, for the treatment
of human cancers.
Thus, the sixth aspect of this invention includes methods of
treating cancer in humans by administering a therapeutically
effective amount of a compound of the first aspect of this
invention, or a pharmaceutical composition of the third aspect of
this invention, to the human. Optionally, the methods further
comprise treating the human with another anticancer therapy, such
as a therapy already conventional for the cancer being treated.
Cancers that are particularly treatable by the method of this
invention are cancers with sensitivity to Aurora kinase inhibitors
and also to inhibitors of angiogenesis (such as to VEGFR2 kinase
inhibitors), and especially those cancers that overexpress one or
more Aurora kinases. Such cancers include those mentioned in the
"Background of the Invention" section above and the documents cited
therein. Cancers particularly treatable by the method of this
invention include solid malignancies such as colorectal, lung,
breast, ovarian, pancreatic, bladder, brain, gastrointestinal, and
kidney cancers, and hematological malignancies, such as leukemias,
especially ALL and CML, lymphomas, and myelodysplastic
syndrome.
The amount of the compound of the first aspect of this invention
that is administered to the human (either alone or, more usually,
in a composition of the third aspect of this invention) should be a
therapeutically effective amount when used alone or when used in
conjunction with the another anticancer therapy (if the compound of
the first aspect of this invention is administered in conjunction
with another anticancer therapy); and similarly the amount of the
another anticancer therapy that is administered to the human (if
the compound of the first aspect of this invention is administered
in conjunction with another anticancer therapy) should be a
therapeutically effective amount when used in conjunction with the
compound of the first aspect of this invention. However, the
therapeutically effective amount of either the compound of the
first aspect of this invention and the amount of the another
anticancer therapy when administered in combination cancer
chemotherapy may each be less than the amount which would be
therapeutically effective if delivered to the human alone. It is
common in cancer therapy, though, to use the maximum tolerated dose
of the or each therapy, with a reduction only because of common
toxicity of the therapies used or potentiation of the toxicity of
one therapy by another.
The compounds of the first aspect of this invention, or
pharmaceutical compositions of the third aspect of this invention,
are thus used to treat cancer in humans requiring such treatment,
by administering a therapeutically effective amount of the chosen
compound or composition. Therapeutically effective amounts of
compounds of the invention are in the range of 10-10,000
mg/m.sup.2, for example, 30-3000 mg/m.sup.2 or 100-1000 mg/m.sup.2.
Dosing may be at 1-35 day intervals; for example, about 500-1000
mg/m.sup.2 at 1-5 week intervals, especially at 1, 2, 3, or 4 week
intervals, or at higher frequencies including as frequently as
once/day for several (e.g. 5 or 7) days, with the dosing repeated
every 2, 3, or 4 weeks, or constant infusion for a period of 6-72
hours, also with the dosing repeated every 2, 3, or 4 weeks.
Suitable dosages and dose frequencies will be readily determinable
by a person of ordinary skill in the art having regard to that
skill and this disclosure. No unacceptable toxicological effects
are expected when compounds of the invention are administered in
accordance with the present invention.
A person of ordinary skill in the art of cancer therapy will be
able to ascertain a therapeutically effective amount of the
compound of the first aspect of this invention and a
therapeutically effective amount of another anticancer therapy (if
the compound of the first aspect of this invention is administered
as a part of a chemotherapeutic combination) for a given cancer and
stage of disease without undue experimentation and in reliance upon
personal knowledge and the disclosure of this application.
EXAMPLES
The following examples illustrate the preparation of compounds of
this invention, and their activity in predictive in vitro and in
vivo anticancer assays.
Preparative and Synthetic Examples
The compounds of this invention are prepared by conventional
methods of organic chemistry. See, for example, Larock,
"Comprehensive Organic Transformations", Wiley-VCH, New York, N.Y.,
U.S.A. In some cases, protective groups may be introduced and later
removed. Suitable protective groups are described in Greene et al.
"Protective Groups in Organic Synthesis", 2nd ed., 1991, John Wiley
and Sons, New York, N.Y., U.S.A. The compounds of this invention
can be synthesized, generally following the synthetic schemes
illustrated earlier in this application, as shown in the following
examples or by modifying the exemplified syntheses by means known
to those of ordinary skill in the art. Preparative examples refer
to the preparation of intermediates useful in the synthesis of
compounds of this invention; synthesis examples refer to the
synthesis of compounds of this invention. Compound numbers refer to
the table immediately following these examples.
Preparative Example 1
Preparation of N-ethyl-3-(4-morpholinyl)propan-1-amine, a sidechain
intermediate for compound 41A
3-(4-Morpholinyl)propan-1-amine (35.0 g, 243 mmol) was dissolved in
tetrahydrofuran (THF, 250 mL), di-tert-butyl dicarbonate (58.0 g,
266 mmol) and N,N-diisopropylethylamine (DIPEA, 84 mL, 484 mmol)
were added, and the solution was stirred overnight. The solvent was
removed and the residue partitioned between ethyl acetate (EA) and
1.2M hydrochloric acid (300 mL). The pH was raised to 7 with
saturated aqueous sodium bicarbonate and the layers separated. The
aqueous layer was extracted twice with EA (150 mL each), and the EA
layers were combined, washed with brine, and dried over magnesium
sulfate. The solvent was removed, giving tert-butyl
3-(4-morpholinyl)propylcarbamate (39 g). Tent-butyl
3-(4-morpholinyl)propylcarbamate (3.5 g, 14.3 mmol) was dissolved
in THF (30 mL), and sodium hydride (60% dispersion in mineral oil,
1.03 g, 25.8 mmol) was added in portions. After stirring for 5 min,
ethyl iodide (13.4 g, 86.0 mmol) was added and the mixture was
heated to 60.degree. C. for 7 hr then stirred at room temperature
overnight. Water (5 mL) was added and the solvent removed. The
residue was dissolved in EA, and the solution was washed with
brine, dried over magnesium sulfate, and the solvent removed to
give crude tert-butyl ethyl[3-(4-morpholinyl)propyl]carbamate (2.5
g). Purification by silica gel chromatography eluting with 92:8
dichloromethane/methanol (DCM/MeOH) gave purified tert-butyl
ethyl[3-(4-morpholinyl)propyl]carbamate (1.4 g). Purified
tert-butyl ethyl[3-(4-morpholinyl)propyl]carbamate (350 mg, 1.3
mmol) was dissolved in 4.0M hydrogen chloride in dioxane (5 mL, 20
mmol) and stirred for 45 min. Water was added and the solution made
basic with 2M aqueous sodium hydroxide. The product was extracted
into EA, and the EA extract washed with brine and dried over
magnesium sulfate. Removal of the EA gave
N-ethyl-3-(4-morpholinyl)propan-1-amine (50 mg).
Preparative Example 2
Preparation of
4-trifluoromethyl-2-methylthio-6-(pyridin-2-yl)-pyrimidine, an
intermediate to compound 11A by Reaction Scheme 1
To a solution of 4,4,4-trifluoro-1-(pyridin-2-yl)butane-1,3-dione
(1.0 g, 4.6 mmol) in ethanol (10 mL) was added
2-methyl-2-thiopseudourea sulfate (0.64 g, 4.6 mmol), followed by
sodium ethoxide (3.0 mL of 21 wt. % solution in ethanol, 9 mmol).
The mixture was heated at reflux for 9 h, then cooled to room
temperature and extracted with EA. The EA was washed with water and
the solvent removed under reduced pressure. The crude product was
purified by column chromatography, eluting with 49:1
chloroform/MeOH, to give
4-trifluoromethyl-2-methylthio-6-(pyridin-2-yl)pyrimidine (0.21 g,
17% yield) as an off-white solid.
Preparative Example 3
Preparation of
(2-chloro-4-trifluoromethylpyrimidin-5-yl)-(4-methylpiperazin-1-yl)methan-
one, an intermediate to compound 130A by Reaction Scheme 1
N-Methylpiperazine (0.11 mL, 1 mmol) was added to a solution of
2-chloro-4-trifluoromethyl-pyrimidine-5-carbonyl chloride (0.24 g,
1 mmol) in DCM (4 mL) at 0.degree. C. The mixture was stirred at
that temperature for 1 hr, and the solvent was then removed under
vacuum to give
(2-chloro-4-trifluoromethylpyrimidin-5-yl)(4-methylpiperazin-1-yl)me-
thanone as a solid.
Other 2-chloro-substituted pyrimidines, intermediates to compounds
131A to 136A, were similarly prepared.
Preparative Example 4
Preparation of
3,3-bis(methylthio)-2-[(4-trifluoromethylpyrimidin-2-yl)acrylonitrile,
an intermediate in Reaction Scheme 2
tert-Butyl 2-cyano-2-[4-trifluoromethylpyrimidin-2
(1H)-ylidene]acetate. tert-Butyl 2-cyanoacetate (97.45 g, 690 mmol)
was dissolved in anhydrous THF (1 L), and cooled on an ice-bath for
90 min with stirring under nitrogen. A 1M solution of lithium
hexamethyldisilazane in THF (690 mL, 690 mmol) was added dropwise.
The mixture was stirred for an additional 1 hr, then
2-chloro-4-trifluoromethylpyrimidin (105 g, 590 mmol) was added
dropwise. The mixture was then heated to 50.degree. C. for 3 hr
with stirring under nitrogen, allowed to cool, and the solvent
removed under reduced pressure. Hydrochloric acid (1N) was added to
the residue to achieve a pH of 1-2. The precipitated solids were
collected by filtration and dried under vacuum to give tert-butyl
2-cyano-2-[4-trifluoromethylpyrimidin-2(1H)-ylidene]acetate (135 g,
82% yield) as a bright yellow solid, >98% pure by LC/MS.
tert-Butyl
2-cyano-2-[4-trifluoromethylpyrimidin-2(1H)-ylidene]acetate (48 g;
166 mmol) was suspended in 4M hydrogen chloride in dioxane (415 mL,
1.66 mol) and the mixture stirred at room temperature for 6 hr,
then concentrated under reduced pressure to give
2-(4-trifluoromethylpyrimidin-2-yl)acetonitrile (31 g, 100% yield)
as an orange oil, >98% pure by LC/MS. To a stirred solution of
2-(4-trifluoromethylpyrimidin-2-yl)acetonitrile (31.0 g; 166 mmol)
in absolute ethanol (800 mL) was added potassium O-ethylxanthate
(26.6 g; 166 mmol) followed by potassium carbonate (45.8 g; 332
mmol). The mixture was heated to 100.degree. C. for 3 hr, cooled to
room temperature, iodomethane (47.1 g; 332 mmol) added dropwise,
and 1N hydrochloric acid (2 L) added. The resulting mixture was
extracted with DCM (1.5 L), and the DCM layer was washed twice with
brine (1 L each). The solvent was removed under reduced pressure to
give
3,3-bis(methylthio)-2-(4-trifluoromethylpyrimidin-2-yl)acrylonitrile
(36 g, 75% yield) as a light brown solid, >95% pure by
LC/MS.
Preparative Example 5
Preparation of 4-[(1H-imidazol-1-yl)methyl]benzene-1,2-diamine, and
intermediate to compound 90A by Reaction Scheme 1 or 2
Potassium carbonate (1.18 g, 8.54 mmol) was added to a stirred
solution of 4-(bromomethyl)-2-fluoro-1-nitrobenzene (2.0 g, 8.55
mmol) and imidazole (584 mg, 8.58 mmol) in acetonitrile (40 mL),
the mixture stirred at room temperature for 3.5 h, the solvent
removed under reduced pressure, and the residue partitioned between
water and EA. The aqueous layer was extracted three times with EA,
then the combined EA layers were extracted three times with 1M
hydrochloric acid. The pH of the combined aqueous layers was
adjusted to 8 with 5M aqueous sodium hydroxide, and the resulting
milky solution was extracted three times with EA. The EA layers
were combined, washed with brine, and dried over magnesium sulfate.
Filtration and concentration gave
1-(3-fluoro-4-nitrobenzyl)-1H-imidazole (914 mg), which contained
about 15% of a bis-alkylated imidazole byproduct.
1-(3-Fluoro-4-nitrobenzyl)-1H-imidazole (914 mg) was treated with
an ethanolic ammonia solution (2M NH.sub.3, 55 mL) in a sealed tube
heated to 80.degree. C. for 2 days. After cooling, the solvent was
removed to give 5-[(1H-imidazol-1-yl)methyl]-2-nitrobenzeneamine
(1.0 g) as an orange solid.
5-[(1H-Imidazol-1-yl)methyl]-2-nitrobenzeneamine (1.0 g, 4.77 mmol)
was dissolved in a 10% solution of dimethylformamide in ethanol (44
mL), and 10% palladium on carbon (130 mg, 0.12 mmol) was added. The
solution was degassed and stirred under a hydrogen atmosphere using
a balloon for 1 day. Following degassing, the solution was diluted
with MeOH and filtered through diatomaceous earth, giving
4-[(1H-imidazol-1-yl)methyl]benzene-1,2-diamine (1.08 g) as a brown
oil.
Preparative Example 6
Preparation of N-(3,4-diaminophenyl)-2-(1H-imidazol-1-yl)acetamide,
an intermediate to compound 86A by Reaction Scheme 1 or 2
To a solution of 1H-imidazole-1-ylacetic acid (284 mg, 2.25 mmol)
and 2-nitrobenzene-1,4-diamine (324 mg, 2.12 mmol) in acetonitrile
(20 mL) was added
3-diethoxyphosphoryloxy-1,2,3-benzotriazin-4(3H)-one (DEPBT, 632
mg, 2.11 mmol) and triethylamine (600 .mu.L, 4.4 mmol). The mixture
was stirred for 4 d at room temperature. The red precipitate was
collected by filtration and washed with acetonitrile to give
N-(4-amino-3-nitrophenyl)-2-(1H-imidazol-1-yl)acetamide (430 mg,
88% yield), 98% pure. A solution of
N-(4-amino-3-nitrophenyl)-2-(1H-imidazol-1-yl)acetamide (430 mg,
1.65 mmol) in ethanol (20 mL) was hydrogenated at atmospheric
pressure with 10% palladium on carbon (100 mg, 0.09 mmol) at room
temperature overnight. The mixture was filtered, giving a blue
solution. Removal of the solvent under reduced pressure gave
N-(3,4-diaminophenyl)-2-(1H-imidazol-1-yl)acetamide (413 mg) as a
blue semi-solid.
Preparative Example 7
Preparation of N-(3,4-diaminophenyl)-2-(diethylamino)acetamide, an
intermediate to compound 75A by Reaction Scheme 1 or 2
2-Nitrobenzene-1,4-diamine (1.55 g, 10.1 mmol), N,N-diethylglycine
sodium salt (1.61 g, 10.6 mmol) and DEPBT (3.02 g, 10.0 mmol) were
suspended in acetonitrile (75 mL). Triethylamine (2.8 mL, 20.0
mmol) was added and the solution stirred overnight at room
temperature. The brown precipitate which was formed was filtered
and discarded. The filtrate was concentrated and the residue
partitioned between EA and saturated aqueous sodium bicarbonate.
The organic phase was washed with water and then with brine, dried
over sodium sulfate, and the solvent removed under reduced
pressure. The residue was dissolved in ethanol (50 mL) and
hydrogenated at atmospheric pressure with 10% palladium on charcoal
(1.0 g, 0.9 mmol) at room temperature overnight. Filtration and
concentration under reduced pressure gave
N-(3,4-diaminophenyl)-2-(diethylamino)acetamide as a light brown
semi-solid, which solidified and turned blue on standing.
Preparative Example 8
Preparation of 4-(pyridin-3-yl)-benzene-1,2-diamine, an
intermediate to compound 107A by Reaction Scheme 1 or 2
4-Amino-3-nitrophenylboronic acid pinacol ester (1.60 g, 6.06
mmol), 3-bromopyridine (1.10 g, 6.96 mmol), cesium carbonate (3.33
g, 10.22 mmol) and
1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride DCM
complex (491 mg, 0.60 mmol) were suspended in dimethylformamide (20
mL). The solution was degassed by vacuum several times and placed
in an argon atmosphere. It was then heated to 65.degree. C. for 6
hr. After cooling, EA (100 mL) and water (40 mL) were added. When
additional water (50 mL) was added to the organic layer, a
precipitate was formed in the separatory funnel. The biphasic
solution was filtered, and the filtrate was transferred to the
separatory funnel and separated. The organic phase was washed twice
with water (50 mL each), then with brine, and then dried with
sodium sulfate and concentrated under reduced pressure to give
2-nitro-4-(pyridin-3-yl)benzenamine (1.18 g).
2-Nitro-4-(pyridin-3-yl)benzenamine (700 mg, 3.26 mmol) in 1:1
ethanol/EA (40 mL) was hydrogenated at atmospheric pressure with
10% palladium on carbon (90 mg, 0.08 mmol) at room temperature for
2 days. Filtration and concentration under reduced pressure gave
4-(pyridin-3-yl)-benzene-1,2-diamine (621 mg).
Preparative Example 9
Preparation of
N-(3,4-diaminophenyl)-2-(diethylamino)-N-ethylacetamide, an
intermediate to compound 139A by Reaction Scheme 1 or 2
A solution of 2-nitrobenzene-1,4-diamine (2.00 g; 13.1 mmol),
di-tert-butyl dicarbonate (3.14 g, 14.4 mmol), and DIPEA (2.6 mL;
14.9 mmol) in dioxane (40 mL) was heated at reflux for 40 min, then
allowed to cool to room temperature. The mixture was concentrated
under reduced pressure and taken up in EA (100 mL). The EA solution
was washed with water and the aqueous phase extracted with EA. The
combined organic layers were washed with saturated aqueous sodium
bicarbonate and brine, dried over magnesium sulfate, and
concentrated under reduced pressure to give tert-butyl
3-amino-4-nitrophenylcarbamate in quantitative yield as a brown
solid. To a stirred solution of tert-butyl
3-amino-4-nitrophenylcarbamate (2.74 g; 10.8 mmol) in anhydrous THF
(50 mL) was added sodium hydride (0.868 g of a 60 wt % dispersion
in oil, 21.7 mmol) in portions over 10 min. The mixture was stirred
for a further 5 min at room temperature and then iodoethane (950
.mu.L; 11.9 mmol) was added. The solution was stirred for 20 hr,
water (1 mL) was added, and the solution concentrated under reduced
pressure. The residue was partitioned between EA and water and the
phases separated. The aqueous phase was extracted twice with EA and
the combined organic phases washed with water, saturated aqueous
sodium bicarbonate and brine. The organic solution was dried over
magnesium sulfate and concentrated under reduced pressure to give a
mixture of mono- and di-ethylated products (2.18 g). This was
purified by silica gel column chromatography eluting with 1:4
EA/hexanes to give tert-butyl 4-amino-3-nitrophenyl(ethyl)carbamate
(0.534 g) as an orange solid. A solution of tert-butyl
4-amino-3-nitrophenyl(ethyl)carbamate (0.514 g; 1.83 mmol) in 4M
hydrogen chloride in dioxane (10 mL) was stirred at ambient
temperature for 3 hr. The solvent was removed under reduced
pressure and the residue dissolved in water. The pH was adjusted to
10 with 1M aqueous sodium hydroxide and the aqueous phase was
extracted three times with EA. The combined organic extracts were
washed with brine, dried over magnesium sulfate, and concentrated
under reduced pressure to give
N.sup.1-ethyl-3-nitrobenzene-1,4-diamine (0.311 g) as a purple
solid. N.sup.1-Ethyl-3-nitrobenzene-1,4-diamine (0.208 g; 1.38
mmol) was dissolved in acetonitrile (10 mL) and N,N-diethylglycine
sodium salt (0.192 g; 1.25 mmol) and DEPBT (0.375 g; 1.25 mmol)
were added, followed by triethylamine (577 .mu.L; 4.14 mmol). The
mixture was stirred at room temperature for 8 hr; and then further
quantities of N,N-diethylglycine sodium salt (0.192 g; 1.25 mmol),
DEPBT (0.375 g; 1.25 mmol), and triethylamine (577 .mu.L; 4.14
mmol) were added and stirring continued for 63 hr. Solids were
removed by filtration and the filtrate concentrated under reduced
pressure. Water was added to the residue and the pH was adjusted to
10 with saturated aqueous sodium bicarbonate. This solution was
extracted three times with EA, and the organic extracts combined,
washed with water and brine, dried over magnesium sulfate, and
concentrated under reduced pressure to give
N-(4-amino-3-nitrophenyl)-2-(diethylamino)-N-ethylacetamide (0.303
g) as a brown oil. A solution of
N-(4-amino-3-nitrophenyl)-2-(diethylamino)-N-ethylacetamide (0.092
g; 0.31 mmol) in ethanol (5 mL) was hydrogenated at atmospheric
pressure with 10% palladium on carbon (11 mg) at room temperature
for 22 hr. The mixture was filtered through diatomaceous earth,
washing with ethanol, and the filtrate was concentrated under
reduced pressure to give
N-(3,4-diaminophenyl)-2-(diethylamino)-N-ethylacetamide in
quantitative yield as a brown oil.
Preparative Example 10
Preparation of 4-(4-methylpiperazin-1-yl)benzene-1,2-diamine, an
intermediate to compound 119A by Reaction Scheme 1 or 2
A solution of 5-fluoro-2-nitrobenzenamine (3.0 g, 18.1 mmol),
triethylamine (5.0 mL, 36.3 mmol), and 1-methylpiperazine (2.0 mL,
18.1 mmol) in dioxane (25 mL) was heated at reflux for 18 hr. After
cooling to room temperature, the mixture was concentrated under
reduced pressure and the residue dissolved in DCM (100 mL). This
solution was washed with saturated aqueous sodium bicarbonate
followed by brine, then dried over magnesium sulfate and
concentrated under reduced pressure to give
5-(4-methylpiperazin-1-yl)-2-nitrobenzenamine (3.25 g) as a dark
brown solid. A solution of
5-(4-methylpiperazin-1-yl)-2-nitrobenzenamine (3.25 g; 13.7 mmol)
in absolute ethanol (40 mL) was hydrogenated at atmospheric
pressure with 10% palladium on carbon (0.30 g; 2.7 mmol) at room
temperature for 17 hr. The mixture was filtered through
diatomaceous earth and concentrated under reduced pressure to give
4-(4-methylpiperazin-1-yl)benzene-1,2-diamine (2.36 g).
Preparative Example 11
Preparation of 4-[2-(1H-imidazol-1-yl)ethoxy]benzenamine, an
intermediate to compound 117A by Reaction Scheme 4
1-(Hydroxyethyl)imidazole (5.30 g; 47.3 mmol) was dissolved in
anhydrous THF (50 mL) and cooled in an ice-bath. Sodium hydride
(2.08 g of a 60 wt % dispersion in oil, 52.0 mmol) was added in
portions over 10 min. The ice-bath was removed, and the mixture was
stirred at room temperature for 20 min. A solution of
1-fluoro-4-nitrobenzene (5.0 mL, 47.2 mmol) in anhydrous THF (10
mL) was added over 5 min and the mixture stirred for a further 1.5
hr. Water (a few mL) was cautiously added and the mixture
concentrated under reduced pressure. The residue was partitioned
between EA (75 mL) and water (75 mL), and the phases separated. The
aqueous phase was extracted twice with EA and the combined organic
layers extracted three times with 1N hydrochloric acid. The pH of
these acidic extracts was then adjusted to 7 with 5N aqueous sodium
hydroxide and the resulting milky solution extracted three times
with EA. The combined organic extracts were washed with water and
brine, then dried over magnesium sulfate and concentrated under
reduced pressure to give 1-(2-(4-nitrophenoxy)-ethyl)-1H-imidazole
(5.46 g) as a brown oil. A solution of
1-(2-(4-nitrophenoxy)ethyl)-1H-imidazole (5.46 g; 2.34 mmol) in
ethanol (60 mL) was hydrogenated at atmospheric pressure with 10%
palladium on carbon (0.40 g) at room temperature for 20 hr. The
mixture was filtered through diatomaceous earth and then
concentrated under reduced pressure to give
4-[2-(1H-imidazol-1-yl)ethoxy]benzenamine (4.56 g) as a white
solid.
4-[3-(4-methylpiperazin-1-yl)propoxy]benzenamine was similarly
prepared using 1-(3-hydroxypropyl)-4-methylpiperazine;
4-[2-(4-methylpiperazin-1-yl)ethoxy]benzenamine was similarly
prepared using 1-(2-hydroxyethyl)-4-methylpiperazine;
4-[2-(4-morpholin-1-yl)ethoxy]benzenamine was similarly prepared
using 4-(2-hydroxyethyl)morpholine; and
4-[2-(dimethylamino)ethoxy]benzenamine was similarly prepared using
2-(dimethylamino)ethanol. Other anilines with oxygen-linked
sidechains may be similarly prepared; in some cases, for example,
the preparation of 4-(pyridin-3-yloxy)-benzenamine from
3-hydroxypyridine and the preparation of
4-[3-(4-morpholin-1-yl)propoxy]-benzenamine from
4-(3-hydroxypropyl)morpholine, less stringent reaction conditions,
such as the use of potassium carbonate as the base, were found to
be sufficient. 3-[2-(4-Morpholin-1-yl)ethoxy]-benzenamine was
prepared using 3-nitrophenol and 4-(2-chloroethyl)morpholine, with
cesium carbonate as the base; and other anilines with oxygen-linked
sidechains may be similarly prepared.
Preparative Example 12
Preparation of 4-[4-(piperidin-1-yl)piperidin-1-yl]benzenamine, an
intermediate to compound 121A by Reaction Scheme 4
A solution of 1-fluoro-4-nitrobenzene (1.3 mL, 12.3 mmol),
triethylamine (6.0 mL; 43.0 mmol) and 4-(piperidin-1-yl)piperidine
(2.36 g; 14.0 mmol) in dioxane (10 mL) was heated at reflux for 18
hr. After cooling to room temperature the mixture was concentrated
under reduced pressure and the residue dissolved in DCM (150 mL).
The DCM solution was washed with saturated aqueous sodium
bicarbonate and brine, then dried over magnesium sulfate and
concentrated under reduced pressure to produce a yellow solid.
Hexanes were added and the suspension briefly sonicated. The solid
was collected by filtration and washed with hexanes to give
1-(4-nitrophenyl)-4-(piperidin-1-yl)piperidine (3.05 g) as a yellow
solid. A solution of 1-(4-nitrophenyl)-4-(piperidin-1-yl)piperidine
(3.05 g; 10.5 mmol) in ethanol (20 mL) was hydrogenated at
atmospheric pressure with 10% palladium on carbon (0.251 g) at room
temperature for 17 hr. The mixture was filtered through
diatomaceous earth and concentrated under reduced pressure to give
4-[4-(piperidin-1-yl)piperidin-1-yl]benzenamine (2.01 g) as a
purple/pink solid.
4-[4-(Morpholin-4-yl)piperidin-1-yl]benzenamine was similarly
prepared from 4-(morpholin-1-yl)piperidine;
4-(4-methylpiperazin-1-yl)benzenamine was similarly prepared from
1-methylpiperazine; and
N.sup.1-methyl-N.sup.1-[2-(piperidine-1-yl)ethyl]benzene-1,4-diamine
was similarly prepared from N-methyl-2-(piperidine-1-yl)ethanamine.
Other anilines with nitrogen-linked sidechains may be similarly
prepared, generally with primary or secondary nitrogens, such as
those on the sidechains of compounds 123A and 124A, protected with
a group such as tert-butoxycarbonyl after the initial coupling with
the 1-fluoro-4-nitrobenzene and the protecting group removed prior
to the final hydrolysis step of the synthesis in which the
resulting anilines are used.
Preparative Example 13
Preparation of 4-[(1H-imidazol-1-yl)methyl]benzenamine, an
intermediate to compound 89A by Reaction Scheme 4
Potassium carbonate (2.00 g; 14.6 mmol) was added to a stirred
solution of 1-(bromomethyl)-4-nitrobenzene (3.20 g; 14.8 mmol) and
imidazole (1.00 g; 14.7 mmol) in acetonitrile (60 mL). The mixture
was stirred at room temperature for 17 hr and then concentrated
under reduced pressure. The residue was partitioned between EA and
water and the phases separated. The aqueous phase was extracted
twice with EA; and the combined organic layers washed with water
and brine, dried over magnesium sulfate, and concentrated under
reduced pressure to give 1-(4-nitrobenzyl)-1H-imidazole (1.27 g) as
a red/brown oil. A solution of 1-(4-nitrobenzyl)-1H-imidazole (2.67
g; 13.1 mmol) in ethanol (100 mL) was hydrogenated at atmospheric
pressure with 10% palladium on carbon (0.285 g) at room temperature
for 4.5 hr. The mixture was filtered through diatomaceous earth and
then concentrated under reduced pressure to give
4-[(1H-imidazol-1-ylmethyl]benzenamine (2.24 g) as a white
solid.
4-[(Morpholin-4-ylmethyl]benzenamine was similarly prepared from
morpholine, and 4-[(4-methylpiperazin-1-ylmethyl]benzenamine was
similarly prepared from 1-methylpiperazine. Other anilines with
methylene-linked sidechains may be similarly prepared, generally
with primary or secondary nitrogens, such as those on the sidechain
of compound 94A, protected with a group such as tert-butoxycarbonyl
after the initial coupling with the 1-fluoro-4-nitrobenzene and the
protecting group removed prior to the final hydrolysis step of the
synthesis in which the resulting anilines are used. Similarly,
other anilines with ethylene-linked sidechains, such as the
sidechain of compound 95A, may be similarly prepared using starting
materials such as 1-(2-bromoethyl)-4-nitrobenzene.
Anilines with other sidechains will be readily prepared by a person
having ordinary skill in the art having regard to that skill and
this disclosure, and will be usable in the syntheses of Reaction
Scheme 4. For example, diethyl (4-nitrobenzyl)phosphonate was
reacted in a Wittig reaction with pyridin-3-carboxaldehyde and the
resulting 3-(4-nitrostyryl)pyridine hydrogenated to give
4-[2-(pyridin-3-yl)ethyl]benzenamine, used to synthesize compound
99A, and the anilines used to synthesize compounds 97A and 98A were
similarly prepared; 4-nitrobenzaldehyde was reacted with glyoxal
and ammonia to give 2-(4-nitrophenyl)-1H-imidazole, which was
N-methylated with methyl iodide and then reduced with stannous
chloride to give 4-(1-methyl-1H-imidazol-2-yl)benzenamine, used to
synthesize compound 106A; 2-fluoro-4-nitrobenzoic acid was
converted to the corresponding benzoyl chloride with oxalyl
chloride, then reacted with 3-(morpholin-4-yl)propan-1-amine and
reduced to give
4-amino-2-fluoro-N-[3-(morpholin-4-yl)propyl]benzamide, used to
synthesize compound 50A; and 4-nitrobenzenesulfonyl chloride was
reacted with 2-(morpholin-4-yl)ethan-1-amine and then reduced with
stannous chloride to give
4-amino-N[2-(morpholin-4-yl)ethyl]benzenesulfonamide, used to
synthesize compound 128A.
Preparative Example 14
Preparation of
(R)--N-(3,4-diaminophenyl)-1-methylpiperidine-2-carboxamide, an
intermediate to compound 143A by Reaction Scheme 2
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU, 4.96 g, 13.1 mmol) was added to a
stirred solution of (R)--N-Boc-2-piperidinecarboxylic acid (3.00 g,
13.1 mmol) and DIPEA (9.0 mL, 51.2 mmol) in anhydrous DMF (50 mL).
The mixture was stirred at room temperature for 5 min and then
2-nitro-1,4-phenylenediamine (3.00 g, 13.1 mmol) was added. The
mixture was stirred for a further 18 hr, then the DMF was removed
by evaporation. The residue was partitioned between water (200 mL)
and EA (100 mL) and the phases separated. The aqueous phase was
extracted with two further portions of EA (100 mL each) and the
combined organic extracts washed with water, 1M sodium carbonate
solution, water, and brine (100 mL each). The solution was dried
over magnesium sulfate and concentrated to give (R)-tert-butyl
2-[(4-amino-3-nitrophenyl)aminocarbonyl]-piperidine-1-carboxylate
as a brown solid (4.80 g).
(R)-tert-Butyl
2-[(4-amino-3-nitrophenyl)aminocarbonyl]piperidine-1-carboxylate
(4.80 g, 13.2 mmol) was suspended in dioxane (100 mL) and hydrogen
chloride (75 mL of a 4M solution in dioxane) was added. The
resulting dark colored solution was stirred at room temperature for
3.5 hr resulting in a yellow/brown suspension. The solid was
collected by filtration and briefly dried under high vacuum. The
solid was suspended in water, the pH was adjusted to pH 8 with 1M
sodium carbonate solution and the resulting suspension stirred for
15 minutes. The solid was collected and dried under high vacuum to
give (R)--N-(4-amino-3-nitrophenyl)piperidine-2-carboxamide
hydrochloride salt as an orange solid (2.08 g). This was treated
with 1M sodium carbonate solution (200 mL) and extracted with EA
(3.times.). The combined EA extracts were washed with brine, dried
over magnesium sulfate, and concentrated to give
(R)--N-(4-amino-3-nitrophenyl)piperidine-2-carboxamide as an orange
foam (1.77 g).
(R)--N-(4-Amino-3-nitrophenyl)piperidine-2-carboxamide (2.09 g, 7.9
mmol) was dissolved in MeOH (50 mL), and paraformaldehyde (0.95 g)
and sodium cyanoborohydride (0.50 g, 8.0 mmol) were added. The
mixture was stirred at room temperature for 70 min, and water added
dropwise. The solvent was evaporated and the residue partitioned
between water and EA (150 mL each). The phases were separated and
the aqueous phase extracted with EA (2.times.100 mL). The combined
EA layers were washed with brine, dried over magnesium sulfate, and
concentrated to give
(R)--N-(4-amino-3-nitrophenyl)-1-methylpiperidine-2-carboxamide as
an orange foam (2.17 g), which was used without purification.
A solution of
(R)--N-(4-amino-3-nitrophenyl)-1-methylpiperidine-2-carboxamide
(2.17 g, 7.8 mmol) in ethanol (75 mL) was degassed under vacuum and
then backfilled with argon. 10% Palladium on carbon (0.44 g) was
added and the mixture again degassed under vacuum. Hydrogen was
introduced by a balloon and the reaction stirred at room
temperature for 23 hr. The mixture was degassed by bubbling argon
through it, and was then filtered through a pad of diatomaceous
earth. The filtrate was concentrated to give
(R)--N-(3,4-diaminophenyl)-1-methylpiperidine-2-carboxamide as a
purple solid (1.78 g).
(S)--N-(3,4-diaminophenyl)-1-methylpiperidine-2-carboxamide, an
intermediate to compound 142A, was prepared by the same method,
starting with (S)--N-Boc-2-piperidinecarboxylic acid.
Preparative Example 15
Preparation of
(S)--N-(3,4-diaminophenyl)-N-ethyl-1-methylpyrrolidine-2-carboxamide,
an intermediate to compound 145A by Reaction Scheme 2
A solution of 2-nitro-1,4-phenylenediamine (10.0 g, 65.3 mmol),
di-tert-butyl dicarbonate (15.7 g, 71.8 mmol) and DIPEA (13.5 mL,
71.8 mmol) in dioxane (150 mL) was heated at gentle reflux for 1 hr
and then allowed to cool to room temperature. The solvent was
evaporated and the residue partitioned between DCM (500 mL) and
water (200 mL) and the phases separated. The aqueous phase was
extracted with DCM (2.times.200 mL) and the combined DCM layers
washed with brine, dried over magnesium sulfate, and concentrated
to give tert-butyl 4-amino-3-nitrophenylcarbamate as a brown solid
(16.6 g).
Sodium hydride (5.24 g of a 60% dispersion in mineral oil, 131
mmol) was added in portions over 25 min to a stirred solution of
tert-butyl 4-amino-3-nitrophenylcarbamate (16.6 g, 65.6 mmol) in
anhydrous THF (200 mL). The mixture was stirred for a further 10
min and iodoethane (5.2 mL, 65.0 mmol) was added. The mixture was
stirred at room temperature for 22 hr and was then quenched by the
addition of a little water. The solvent was evaporated and the
residue partitioned between EA and brine (300 mL each). The phases
were separated and the aqueous phase extracted with EA (2.times.150
mL). The combined EA layers were washed with brine, dried over
magnesium sulfate, and concentrated to give a brown gum (16.0 g),
which was purified by column chromatography eluting with 30% EA,
70% hexanes to give tert-butyl
4-amino-3-nitrophenyl(ethyl)carbamate as a brown foam (3.59 g).
The tert-butyl 4-amino-3-nitrophenyl(ethyl)carbamate from the
previous step was dissolved in dioxane (35 mL) and hydrogen
chloride (35 mL of a 4M solution in dioxane) was added. The mixture
was stirred at room temperature for 18 hr and then the solvent was
evaporated. The residue was dissolved in water (200 mL) and the
solution adjusted to pH10 with 5M sodium hydroxide solution. The
solution was extracted with EA (3.times.100 mL), and the combined
EA extracts were washed with brine, dried over magnesium sulfate,
and concentrated to give N.sup.1-ethyl-3-nitrobenzene-1,4-diamine
as a purple solid (1.68 g).
Triethylamine (1.0 mL, 7.33 mmol) was added to a stirred solution
of N.sup.1-ethyl-3-nitrobenzene-1,4-diamine (440 mg, 2.43 mmol),
N-methyl-L-proline (314 mg, 2.43 mmol) and DEPBT (727 mg, 2.43
mmol) in acetonitrile (15 mL). The mixture was stirred at room
temperature and further aliquots of N-methyl-L-proline (314 mg,
2.43 mmol), DEPBT (727 mg, 2.43 mmol), and triethylamine (1.0 mL,
7.33 mmol) were added after 1, 2, and 3 days. After stirring for a
further 1 day, the mixture was concentrated and the residue taken
up in EA (75 mL). The EA solution was extracted with water, and
with 1M sodium carbonate solution (2.times.). The combined water
and 1M sodium carbonate solution extracts were back-extracted with
EA (4.times.) and the combined EA extracts washed with brine, dried
over magnesium sulfate, and concentrated to give
(S)--N-(4-amino-3-nitrophenyl)-N-ethyl-1-methyl-pyrrolidine-2-carboxamide
(388 mg) as an orange/brown oil. This was hydrogenated in ethanol,
using 10% palladium on carbon (153 mg) as the catalyst. After
stirring overnight under a hydrogen atmosphere (balloon), the
mixture was filtered through a pad of diatomaceous earth and the
filtrate concentrated to give
(S)--N-(3,4-diaminophenyl)-N-ethyl-1-methylpyrrolidine-2-carboxamide
(335 mg).
Synthesis Example 1
Synthesis of
2-[1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethyl-pyrimidin-2-yl)ace-
tamide, compound 1A, by Reaction Scheme 1
A solution of 2-[1H-benzimidazol-2(3H)-yl]acetonitrile (10 g, 64
mmol), di-tert-butyl dicarbonate (16.6 g, 76 mmol), and
triethylamine (8.8 mL, 64 mmol) in THF (180 mL) was stirred at room
temperature overnight. The solvent was removed under vacuum, and
the crude product was dissolved in chloroform and washed with
water, 1N hydrochloric acid, and saturated aqueous sodium
bicarbonate. The chloroform phase was dried over magnesium sulfate,
and the solvent was removed under vacuum to give
2-[1-(tert-butoxycarbonyl)benzimidazol-2(3H)-yl]acetonitrile (15 g)
as a solid.
A solution of
2-[1-(tert-butoxycarbonyl)benzimidazol-2(3H)-yl]acetonitrile (6 g,
23 mmol) in THF (50 mL) was added to a suspension of 60% sodium
hydride in mineral oil (1.38 g, 35 mmol) in tetrahydrofuran (85 mL)
at 0.degree. C. over 10 min. The suspension was stirred at
0.degree. C. for 15 min, and 2-chloro-4-trifluoromethylpyrimidin
(2.79 mL, 23 mmol) was added. The mixture was allowed to warm to
room temperature and stirred overnight, and water (300 mL) and then
1N hydrochloric acid (40 mL) were added. The crude product was
extracted into chloroform and washed twice with water (100 mL
each). The organic phase was dried over magnesium sulfate, and the
solvent was removed under vacuum to give
2-[1-(tert-butoxycarbonyl)benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethy-
lpyrimidin-2-yl)acetonitrile (8.17 g) as a brown oil.
2-[1-(tert-Butoxycarbonyl)benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethy-
lpyrimidin-2-yl)-acetonitrile (8.1 g, 20 mmol) was dissolved in
concentrated sulfuric acid (30 mL), and the solution added
gradually to ice/water (100 mL), allowed to warm to room
temperature, and stirred overnight. The mixture was cooled to
0.degree. C., poured over ice, and neutralized with 50% aqueous
sodium hydroxide. Water was added, and the crude product was
extracted with 4:1 chloroform/isopropanol. The organic phase was
dried over magnesium sulfate, the solvent was removed under vacuum,
and the solid was purified by reverse phase preparative HPLC to
give
2-[1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-yl-
)acetamide (1.18 g, 18% overall yield) as a yellow solid.
Compounds 4A, 132A, 135A, and 138A, for example, were prepared by
this general method, using the appropriately substituted starting
materials.
Synthesis Example 2
Synthesis of
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide,
compound 2A, by Reaction Scheme 1
2-(Benzothiazol-2-yl)acetonitrile (0.69 g, 4 mmol) was added in
portions to a suspension of 95% sodium hydride ((0.21 g, 6 mmol) in
THF (12 mL). The resulting yellow suspension was stirred at room
temperature for 30 min, and 2-chloropyrimidine (0.45 g, 4 mmol) was
added. The mixture was stirred for 3 d, and water was added to the
dark solution. The mixture was acidified with 1N hydrochloric acid,
and the precipitate that formed was collected by filtration, washed
with water and acetonitrile, and dried under vacuum to give
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile as a
brown solid.
2-[Benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetonitrile (0.1
g, 0.39 mmol) was dissolved in concentrated sulfuric acid (2 mL)
and stirred at room temperature for 5 hr. The mixture was cooled to
0.degree. C., poured over ice, and neutralized with 50% aqueous
sodium hydroxide. The precipitate that formed was collected by
filtration and purified by reverse phase preparative HPLC to give
2-[benzothiazol-2(3H)-ylidene]-2-(pyrimidin-2-yl)acetamide (42 mg,
40% yield) as a yellow solid.
Compounds 3A, 16A, 18A, 21A, 22A, and 23A, for example, were
prepared by this general method, using the appropriately
substituted starting materials. Compound 5A was prepared from the
nitrile intermediate to compound 3A by hydrolysis of a methanolic
solution of the intermediate with 1M aqueous lithium hydroxide for
2 d at room temperature, neutralization with 1N hydrochloric acid,
collection of the precipitate by filtration, washing with water and
acetonitrile, and drying to give
2-[benzothiazol-2(3H)-ylidene]-2-(5-carboxy-4-trifluoromethylpyrimidin-2--
yl)acetonitrile. This was hydrolyzed with concentrated sulfuric
acid and purified to give
2-[benzothiazol-2(3H)-ylidene]-2-(5-carboxy-4-trifluoromethylpyrimidin-2--
yl)acetamide, compound 5A. Compound 24A was similarly prepared from
the nitrile intermediate to compound 23A.
Synthesis Example 3
Synthesis of
2-[benzothiazol-2(3H)-ylidene]-2-[5-(4-methylpiperazine-1-carbonyl)-4-tri-
fluoromethylpyrimidin-2-yl]acetamide, compound 130A, by Reaction
Scheme 1
2-(Benzothiazol-2-yl)acetonitrile (2.05 g, 12 mmol) was added in
portions to stirred concentrated sulfuric acid (5 mL) over 10 min.
The mixture was stirred for 4 hr, then poured over ice/water. The
precipitate that formed was extracted into EA and washed with
brine, then the organic phase was dried over magnesium sulfate and
the solvent removed under vacuum to give
2-(benzothiazol-2-yl)acetamide (1.02 g) as an orange solid.
Sodium hydride 99% (50.8 mg, 2.1 mmol) was added to a suspension of
2-(benzothiazol-2-yl)acetamide (103 mg, 0.53 mmol) in THF (2 mL),
and the resulting yellow suspension was stirred at room temperature
for 15 min. The mixture was cooled to 0.degree. C. and
(2-chloro-4-trifluoromethyl-pyrimidin-5-yl)(4-methylpiperazin-1-yl)methan-
one (0.18 g, 0.53 mmol) was added. The mixture was stirred at room
temperature overnight and the solvent was then removed under
reduced pressure. The residue was suspended in water and the pH
adjusted to 7 with 1N hydrochloric acid. The solid was collected by
filtration and partitioned between EA and water. The EA phase was
separated, washed with brine, dried over magnesium sulfate, and
concentrated under vacuum. The resulting material was purified by
reverse phase preparative HPLC to give
2-[benzothiazol-2(3H)-ylidene]-2-[5-(4-methylpiperazine-1-carbonyl)-4-tri-
fluoromethylpyrimidin-2-yl]acetamide hydrochloride (87 mg, 33%
yield) as a yellow solid.
Compounds 2A, 131A, and 134A, for example, were prepared by this
general method using the appropriately substituted starting
materials.
Synthesis Example 4
Synthesis of
2-(5-{[3-(4-morpholinyl)propyl]aminocarbonyl}-1H-benzimidazol-2(3H)-ylide-
ne)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, compound 39A, by
Reaction Scheme 2
A mixture of
3,3-bis(methylthio)-2-(4-trifluoromethylpyrimidin-2-yl)acrylonitrile
(682 mg, 2.34 mmol), 3,4-diaminobenzoic acid (392 mg, 2.57 mmol,
1.1 eq.), and 4-dimethylaminopyridine (143 mg, 1.17 mmol, 0.5 eq.)
in ethanol (3 mL) was heated at 150.degree. C. for 0.5 hr in a
microwave. The mixture was cooled to room temperature, concentrated
hydrochloric acid (1 mL) was added, and the mixture was allowed to
stand for 0.5 hr. The precipitate that formed was collected by
filtration, washed with ethanol and acetonitrile, and dried under
vacuum to give
2-[5-carboxy-1H-benzimidazol-2(3H)-ylidene]-2-(3-trifluoromethylpyrimidin-
-2-yl)acetonitrile (450 mg, 1.29 mmol, 55% yield) as a brown
solid.
The
2-[5-carboxy-1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrim-
idin-2-yl)-acetonitrile (450 mg, 1.29 mmol) from the previous step,
dissolved in concentrated sulfuric acid (2 mL), was heated to
50.degree. C. for 6 hr. The mixture was cooled to room temperature
and added dropwise to rapidly stirred room-temperature water (500
mL). The precipitate that formed was collected by filtration,
washed with water, and dried under vacuum to give
2-[5-carboxy-1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-
-2-yl)acetamide (322 mg, 0.88 mmol, 68% yield) as a dark brown
solid.
To a room temperature solution of
2-[5-carboxy-1H-benzimidazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-
-2-yl)acetamide (630 mg, 1.73 mmol) in dimethylformamide (20 mL)
was added DIPEA (661 .mu.L, 3.79 mmol, 2.2 eq.), HBTU (720 mg, 1.90
mmol, 1.1 eq.). After 5 min, 3-(4-morpholinyl)propan-1-amine (277
.mu.L, 1.90 mmol, 1.1 eq.) was added and the mixture was stirred at
room temperature for 3 hr. The mixture was diluted with EA (50 mL)
and washed three times with 5% aqueous sodium bicarbonate (50 mL
each). The EA fraction was decanted, dried over magnesium sulfate,
filtered, and concentrated under vacuum to give a yellow solid.
About 2 mL of 4.0 M hydrogen chloride in dioxane was added, and the
mixture was stirred for 15 min and then concentrated under vacuum
to give
2-(5-{[3-(4-morpholinyl)propyl]aminocarbonyl}-1H-benzimidazol-2(3H)-ylide-
ne)-2-(4-trifluoromethylpyrimidin-2-yl)acetamide hydrochloride (750
mg, 1.52 mmol, 88% yield) as a yellow solid.
Compounds 33A, 34A, 41A, 44A, 45A, 48A, 51A, 53A, 73A, 77A, 79A to
83A, 86A, and 142A to 146A, for example, were prepared by this
general method using the appropriately substituted starting
materials.
Synthesis Example 5
Synthesis of
2-(6-{[3-(4-morpholinyl)propyl]aminocarbonyl}-benzothiazol-2(3H)-ylidene)-
-2-(4-trifluoromethylpyrimidin-2-yl)acetamide, compound 40A, by
Reaction Scheme 2
A mixture of 4-amino-3-mercaptobenzoic acid (768 mg, 4.5 mmol),
3,3-bis(methylthio)-2-(4-trifluoromethylpyrimidin-2-yl)acrylonitrile
(1.45 g, 5.0 mmol, 1.1 eq.), 4-dimethylaminopyridine (550 mg, 4.5
mmol, 1 eq.), and potassium carbonate (622 mg, 4.5 mmol, 1 eq.) in
ethanol (20 mL) was heated at 160.degree. C. for 0.4 hr in a
microwave. The mixture was cooled to room temperature, diluted with
DCM, and washed with 1M hydrochloric acid. The DCM fraction was
decanted, filtered, and concentrated under vacuum to give
2-[6-carboxybenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethyl-pyrimidin-2--
yl)acetonitrile (729 mg, 40% yield) as a brown solid.
A solution of
2-[6-carboxybenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-y-
l)-acetonitrile (620 mg, 1.70 mmol) in concentrated sulfuric acid
(5 mL), was heated to 50.degree. C. for 10 hr. The mixture was
cooled to room temperature and added dropwise to rapidly stirred
room-temperature water (100 mL). The precipitate that formed was
collected by filtration, washed with water and hexanes, and dried
under vacuum to give
2-[6-carboxybenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-y-
l)acetamide (587 mg, 90% yield) as a brown solid.
To a room temperature solution of
2-[6-carboxybenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-y-
l)acetamide (350 mg, 0.92 mmol) in acetonitrile (5 mL) was added
triethylamine (250 .mu.L, 1.79 mmol, 2 eq.), DEPBT (299 mg, 1.0
mmol, 1.1 eq.), and 3-(4-morpholinyl)-propan-1-amine (134 .mu.L,
0.92 mmol, 1.0 eq.), and the mixture was stirred at room
temperature until a yellow-brown precipitate formed. The
precipitate was collected by filtration, washed with acetonitrile,
and dried under vacuum to give a yellow solid. About 2 mL of 4.0 M
hydrogen chloride in dioxane was added, and the mixture was stirred
for 15 min and then concentrated under reduced pressure to give
2-(6-{[3-(morpholin-4-yl)propyl]aminocarbonyl}-benzothiazol-2(3H)-ylidene-
)-2-(4-trifluoromethyl-pyrimidin-2-yl)acetamide hydrochloride (340
mg, 73% yield) as a yellow solid.
Synthesis Example 6
Synthesis of
2-[6-nitrobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethyl-pyrimidin-2-yl-
)acetamide, compound 26A, by Reaction Scheme 3
To a solution of 2-(methylthio)-6-nitro-1,3-benzothiazole (226 mg,
1.0 mmol) in ethanol (3.5 mL) was added
2-(4-trifluoromethylpyrimidin-2-yl)acetonitrile (187 mg, 1.0 mmol)
and potassium carbonate (207 mg, 1.5 mmol). The orange mixture was
heated in the microwave at 160.degree. C. for 15 min, then cooled
and poured into EA (60 mL). The organic layer was washed twice with
1N hydrochloric acid (25 mL each), brine (25 mL), and water (5 mL),
then dried with magnesium sulfate, filtered, and concentrated under
reduced pressure to give
2-(6-nitrobenzothiazol-2(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)-
acetonitrile (349 mg, 96% yield) as a yellow solid.
2-(6-Nitrobenzothiazol-2(3H)-ylidene)-2-(4-trifluoromethylpyrimidin-2-yl)-
acetonitrile (296 mg, 0.81 mmol) was dissolved in concentrated
sulfuric acid (3.0 mL). The reddish mixture was stirred at
50.degree. C. for 8 hr, then poured into cold water (25 mL). The
yellow precipitate was collected by filtration, washed with hexanes
(30 mL), and dried under vacuum to give
2-[6-nitrobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin--
2-yl)acetamide (280 mg, 89% yield) as an orange solid.
Compound 32A was prepared from compound 26A as follows. To a
solution of
2-[6-nitrobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-yl)-
acetamide (280 mg, 0.73 mmol) in MeOH (25 mL) was added a slurry of
10% palladium on carbon (200 mg) in MeOH (10 mL), and the mixture
was stirred under hydrogen at atmospheric pressure for 24 hr. The
mixture was filtered and the filtrate concentrated to give crude
2-[6-aminobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-yl)-
acetamide as a red powder. This was purified by flash column
chromatography eluting with 19:1 EA/MeOH to give pure
2-[6-aminobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-yl)-
acetamide, compound 32A (160 mg, 62% yield) as a yellow powder.
Compound 76A was prepared from compound 32A as follows. To a
solution of N-(tert-butoxycarbonyl)glycine hydrochloride (20 mg) in
DMF (5 mL) were added
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (190 mg), triethylamine (388 .mu.L, 3.88 mmol),
and
2-[6-aminobenzothiazol-2(3H)-ylidene]-2-(4-trifluoromethylpyrimidin-2-yl)-
acetamide (141 mg). The mixture was stirred at room temperature for
1 hr, then poured into EA (50 mL). The EA layer was washed three
times with 10% aqueous sodium bicarbonate (30 mL each), and three
times with water (15 mL each), then dried over magnesium sulfate,
filtered, and concentrated under reduced pressure to give
2-{6-[(tert-butyloxycarbonylamino)acetylamino]benzothiazol-2(3H)-ylidene}-
-2-(4-trifluoromethylpyrimidin-2-yl)acetamide as a brown foam.
2-{6-[(Tert-butyloxycarbonylamino)-acetylamino]benzothiazol-2(3H)-ylidene-
}-2-(4-trifluoromethylpyrimidin-2-yl)acetamide (80 mg, 156 mmol)
was dissolved in 4N hydrogen chloride in dioxane (5.0 mL, 20 mmol),
and the mixture stirred at room temperature for 2 hr. The mixture
was concentrated to give
2-{6-[(2-aminoacetyl)-amino]benzothiazol-2(3H)-ylidene}-2-(4-trifluoromet-
hylpyrimidin-2-yl)acetamide (50 mg) as a beige solid. Other
compounds with an amide side-chain may be similarly prepared from
compound 32A.
Synthesis Example 7
Synthesis of
2-{6-[2-(1H-imidazol-1-yl)ethoxy]benzothiazol-2(3H)-ylidene}-2-(4-trifluo-
romethylpyrimidin-2-yl)acetamide, compound 117A, by Reaction Scheme
4
To an ice-cooled stirred biphasic solution of
4-[2-(1H-imidazol-1-yl)ethoxy]benzenamine (1.00 g; 4.92 mmol) and
sodium bicarbonate (2.07 g; 24.6 mmol) in chloroform (100 mL) and
water (50 mL) was added a solution of thiophosgene (452 .mu.L; 5.90
mmol) in chloroform (10 mL) over 5 min. The mixture was stirred at
ice-bath temperature for 20 min, and then the aqueous and organic
phases were separated and the aqueous phase extracted with
chloroform (50 mL). The combined chloroform layers were washed with
brine, then dried over magnesium sulfate and concentrated under
reduced pressure to give
1-[2-(4-isothiocyanatophenoxy)ethyl]-1H-imidazole (1.41 g) as a
yellow oil.
Sodium hydride (0.531 g of a 60 wt % dispersion in oil, 13.3 mmol)
was added in portions to a stirred solution of
2-(4-trifluoromethylpyrimidin-2-yl)acetamide (0.911 g; 4.43 mmol)
in THF (20 mL) over 10 min. A solution of
1-[2-(4-isothiocyanatophenoxy)ethyl]-1H-imidazole (1.21 g; 4.92
mmol) in THF (10 mL) was then added over 15 min. The mixture was
stirred at room temperature for a further 1 hr, and then water (1
mL) was added and the mixture was concentrated under reduced
pressure. Water (75 mL) was added to the residue, the pH adjusted
to 7 with 2N hydrochloric acid, and the mixture was extracted three
times with EA. The combined EA extracts were washed with brine,
dried over magnesium sulfate, and concentrated under reduced
pressure to give
3-{-4-[2-(1H-imidazol-1-yl)ethoxy]phenylamino}-3-thioxo-2-(4-trifluoromet-
hylpyrimidin-2-yl)propanamide (1.47 g) as an orange semi-solid.
A solution of
3-{4-[2-(1H-imidazol-1-yl)ethoxy]phenylamino}-3-thioxo-2-(4-trifluorometh-
yl-pyrimidin-2-yl)propanamide (1.47 g, 3.29 mmol) in glacial acetic
acid (10 mL) was cooled in a cold water bath and a solution of
bromine (126 .mu.L, 2.46 mmol) in glacial acetic acid (1 mL) added
dropwise over 10 min. The mixture was allowed to warm to room
temperature with stirring for a further 35 min, and the acetic acid
was decanted. The residue was dried under vacuum, then dissolved in
acetonitrile/water and purified by reverse phase preparative HPLC
to give produce
2-{6-[2-(1H-imidazol-1-yl)ethoxy]-benzothiazol-2(3H)-ylidene}-2-(4-triflu-
oromethylpyrimidin-2-yl)acetamide hydrochloride (0.406 g) as an
orange solid.
Compounds 50A, 52A, 89A, 91A, 93A to 95A, 97A to 101A, 103A, 104A,
106A, 108A, 110A to 116A, 118A, and 120A to 129A, for example, were
prepared by this general method using the appropriately substituted
starting materials.
Synthesis Example 8
Synthesis of
2-[benzothiazol-2(3H)-ylidene-2-[4-tert-butyl-6-(trifluoromethyl)pyrimidi-
n-2-yl]acetamide, compound 7A, by Reaction Scheme 5
To a stirred solution of 2-chlorobenzothiazole (1.0 mL, 8.1 mmol)
and malononitrile (534 mg; 8.1 mmol) in acetonitrile (4 mL) was
added sodium ethoxide (3.0 mL of 21 wt % solution in ethanol, 8.0
mmol). The mixture was stirred at room temperature for 4 d, then
acidified with 2M hydrochloric acid. The solid was collected by
filtration, washed with acetonitrile, and dried under vacuum to
give 2-(benzothiazol-2(3H)-ylidene)malononitrile (770 mg) as a
white solid.
A suspension of 2-(benzothiazol-2(3H)-ylidene)malononitrile (0.45
g, 2.25 mmol) in dioxane (10 mL) and ethanol (10 mL) was cooled to
0.degree. C. and hydrogen chloride gas was bubbled into the mixture
for 10 min. The mixture was allowed to warm to room temperature,
then heated to 50.degree. C. overnight. The resulting precipitate
was collected by filtration, washed with acetonitrile, and dried
under vacuum to give
2-[benzothiazol-2(3H)-ylidene]-2-cyanoacetimidate (0.43 g) as a
white solid.
A suspension of 2-[benzothiazol-2(3H)-ylidene]-2-cyanoacetimidate
(0.4 g, 1.6 mmol) in ethanol (5 mL) was placed in a pressure tube,
cooled to -78.degree. C., and gaseous ammonia was bubbled into the
tube. The mixture was allowed to warm to room temperature and
stirred for 3 d. The solvent was removed under vacuum to give
2-[benzothiazol-2(3H)-ylidene]-2-cyanoacetamidine as a beige
solid.
2-[Benzothiazol-2(3H)-ylidene]-2-cyanoacetamidine (0.2 g, 0.92
mmol) was dissolved in dimethylsulfoxide (1 mL), and
1,1,1-trifluoro-5,5,-dimethyl-2,4-hexanedione (320 .mu.L, 1.84
mmol) and sodium ethoxide (0.6 mL of 21 wt % solution in ethanol,
1.84 mmol) were added. The solution was heated to 180.degree. C.
under microwave irradiation for 20 min, dissolved in EA, and washed
with 1N hydrochloric acid and then water. Removal of the solvent
gave
2-[benzothiazol-2(3H)-ylidene]-2-[4-tert-butyl-6-(trifluoromethyl)pyrimid-
in-2-yl]acetonitrile as a beige solid.
2-[Benzothiazol-2(3H)-ylidene]-2-[4-tert-butyl-6-(trifluoromethyl)pyrimid-
in-2-yl]acetonitrile (150 mg, 0.39 mmol) was dissolved in
concentrated sulfuric acid (1.5 mL) and stirred at room temperature
overnight. The mixture was cooled to 0.degree. C., poured over ice,
and neutralized with 50% aqueous sodium hydroxide. The product was
partially extracted into chloroform, and dried to a brown solid (57
mg), which was purified by reverse phase preparative HPLC to give
2-[benzothiazol-2(3H)-ylidene]-2-[4-tert-butyl-6-(trifluoromethyl)pyrimid-
in-2-yl]acetamide (13 mg) as a yellow solid.
Synthesis Example 9
Synthesis of
2-{5-[(S)-(1-methylpyrrolidin-2-yl)carbonylamino]-1H-benzimidazol-2(3H)-y-
lidene}-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetamide,
compound 144A
A solution of 1,1,1-trifluoro-2,4-pentanedione (5.00 g; 32.4 mmol)
and S-methylisothiourea hemisulfate (4.06 g; 29.2 mmol) in pyridine
(3.75 mL) and water (62 mL) was heated at reflux for 1 day. The
mixture was cooled to room temperature, extracted with chloroform,
and the chloroform extract washed with water to give
4-methyl-2-(methylthio)-6-(trifluoromethyl)pyrimidine as an
off-white solid (4.60 g, 22.1 mmol). This was dissolved in MeOH (60
mL), and a solution of OXONE.RTM. (27.1 g, 44.2 mmol) in water (100
mL) was added over 10 min. The mixture was stirred at room
temperature for 4 hr and then extracted with EA. The EA was washed
with water, dried, and concentrated to give
4-methyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine (4.8 g)
as a white solid. Sodium hydride (0.16 g of a 60% dispersion in
mineral oil; 4.0 mmol) was added to a stirred solution of ethyl
cyanoacetate (0.21 mL; 2.0 mmol) in THF (8 mL), and the resulting
suspension stirred at room temperature for 15 min.
4-Methyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine (0.48 g;
2.0 mmol) was then added, and the mixture stirred overnight. Water
was added, followed by 1M hydrochloric acid to adjust to pH 3-4.
The solid that formed solid was filtered, washed with water, and
dried under high vacuum to give ethyl
2-cyano-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetate
hydrochloride salt (0.43 g) as a pale yellow solid.
4-Nitro-1,2-phenylenediamine (50 mg; 0.32 mmol) and ethyl
2-cyano-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetate
hydrochloride salt (101 mg; 0.32 mmol) were heated at 198.degree.
C. in a microwave for 3 min. Acetic acid (1.5 mL) was added to the
crude material and the mixture heated at 100.degree. C. for 6 hr.
The solid was collected by filtration, washed with acetonitrile,
and dried under high vacuum to give
2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]-2-(5-nitro-1H-benzimidazol-
-2(3H)-ylidene)acetonitrile (59 mg) as a yellow solid.
2-[4-Methyl-6-(trifluoromethyl)pyrimidin-2-yl]-2-(5-nitro-1H-benzimidazol-
-2(3H)-ylidene)acetonitrile (55 mg; 0.15 mmol) in DMF (3 mL) was
hydrogenated over 10% palladium on carbon (11 mg). The mixture was
filtered through diatomaceous earth, washing with methanol, and the
filtrate was concentrated to give
2-(5-amino-1H-benzimidazol-2(3H)-ylidene)-2-[4-methyl-6-(trifluoromethyl)-
pyrimidin-2-yl]acetonitrile, which was dissolved in DMF (1 mL) and
added to a solution of N-methyl-L-proline (19.3 mg; 0.16 mmol),
HBTU (56.8 mg; 0.15 mmol), and DIPEA (104.5 .mu.L; 0.60 mmol) in
DMF (1 mL). The mixture was stirred for 5 hr and then partitioned
between EA and water. The EA phase was dried and concentrated to
give
2-{5-[(S)-(1-methylpyrrolidin-2-yl)carbonylamino]-1H-benzimidazol-2
(3H)-ylidene}-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetonitrile
(65 mg) as a brown oil.
2-{5-[(S)-(1-Methylpyrrolidin-2-yl)-carbonylamino]-1H-benzimidazol-2(3H)--
ylidene}-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]-acetonitrile
(65 mg) was dissolved in concentrated sulfuric acid and stirred at
room temperature overnight. The mixture was diluted with ice-water
and acetonitrile and, after coming to room temperature, was
purified by reverse phase preparative HPLC using a Peeke Ultro 120,
7 .mu.m, C18Q, 250.times.30 mm column at a flow rate of 42 mL/min
and mobile phases of 95% water, 5% acetonitrile (with 0.01%
hydrochloric acid) and 5% water, 95% acetonitrile (with 0.01%
hydrochloric acid) to give
2-{5-[(S)-(1-methylpyrrolidin-2-yl)carbonylamino]-1H-benzimidazol-2
(3H)-ylidene}-2-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]acetamide
hydrochloride salt (18 mg) as a yellow solid.
The compounds of formula A as shown in the table below were
prepared by one or more of the above methods, or similar methods
not described in detail here. All of the compounds of formula A
were analyzed to confirm identity and purity, using HPLC for
purity, and one or more of mass spectrometry (using either positive
or negative ionization) and NMR (.sup.1H and/or .sup.13C) for
identity, and were confirmed to be the expected product in good
purity. Other compounds of formula A may be similarly prepared.
Representative compounds of formula A include ("exact mass" is of
the parent compound; mass spectra were with positive ionization and
mass M+H, unless the mass was noted otherwise):
TABLE-US-00001 Compound Structure Exact mass, M MS (m/z) 1A
##STR00009## 321 322 2A ##STR00010## 338 339 3A ##STR00011## 396
397 4A ##STR00012## 379 380 5A ##STR00013## 382 383 6A ##STR00014##
368 369 7A ##STR00015## 394 395 8A ##STR00016## 352 353 9A
##STR00017## 406 407 10A ##STR00018## 415 414 (M - H) 11A
##STR00019## 415 414 (M - H) 12A ##STR00020## 336 337 13A
##STR00021## 353 354 14A ##STR00022## 467 468 15A ##STR00023## 466
467 16A ##STR00024## 300 301 17A ##STR00025## 283 284 18A
##STR00026## 284 285 19A ##STR00027## 267 268 20A ##STR00028## 346
347 21A ##STR00029## 298 299 22A ##STR00030## 298 299 23A
##STR00031## 342 343 24A ##STR00032## 328 327 (M - H) 25A
##STR00033## 464 463 (M - H) 26A ##STR00034## 383 384 27A
##STR00035## 382 383 28A ##STR00036## 365 366 29A ##STR00037## 379
380 30A ##STR00038## 452 453 31A ##STR00039## 453 454 32A
##STR00040## 353 354 33A ##STR00041## 404 405 34A ##STR00042## 419
420 35A ##STR00043## 351 352 36A ##STR00044## 357 358 37A
##STR00045## 373 374 38A ##STR00046## 357 358 39A ##STR00047## 491
492 40A ##STR00048## 508 509 41A ##STR00049## 519 520 42A
##STR00050## 536 537 43A ##STR00051## 469 470 44A ##STR00052## 441
443 (M + 2) 45A ##STR00053## 472 473 46A ##STR00054## 489 490 47A
##STR00055## 475 474 (M - H) 48A ##STR00056## 500 501 49A
##STR00057## 517 518 50A ##STR00058## 526 527 51A ##STR00059## 511
512 52A ##STR00060## 508 509 53A ##STR00061## 477 478 54A
##STR00062## 494 495 55A ##STR00063## 518 519 56A ##STR00064## 535
536 57A ##STR00065## 507 508 58A ##STR00066## 447 448 59A
##STR00067## 464 465 60A ##STR00068## 433 434 61A ##STR00069## 450
451 62A ##STR00070## 449 451 63A ##STR00071## 424 425 64A
##STR00072## 421 422 65A ##STR00073## 438 439 66A ##STR00074## 447
448 67A ##STR00075## 464 465 68A ##STR00076## 433 434 69A
##STR00077## 450 451 70A ##STR00078## 447 448 71A ##STR00079## 464
465 72A ##STR00080## 540 540 73A ##STR00081## 421 422 74A
##STR00082## 438 439 75A ##STR00083## 449 450 76A ##STR00084## 410
411 77A ##STR00085## 447 448 78A ##STR00086## 464 465 79A
##STR00087## 433 434 80A ##STR00088## 433 434 81A ##STR00089## 421
422 82A ##STR00090## 461 462 83A ##STR00091## 490 491 84A
##STR00092## 477 478 85A ##STR00093## 491 492 86A ##STR00094## 444
445 87A ##STR00095## 458 459 88A ##STR00096## 461 462 89A
##STR00097## 418 419 90A ##STR00098## 401 402 91A ##STR00099## 437
438 92A ##STR00100## 399 400 93A ##STR00101## 450 451 94A
##STR00102## 436 437 95A ##STR00103## 432 433 96A ##STR00104## 415
416 97A ##STR00105## 446 447 98A ##STR00106## 446 447 99A
##STR00107## 443 444 100A ##STR00108## 499 500 101A ##STR00109##
451 452 102A ##STR00110## 434 435 103A ##STR00111## 467 468 104A
##STR00112## 485 486 105A ##STR00113## 365 366 106A ##STR00114##
418 419 107A ##STR00115## 398 399 108A ##STR00116## 415 416 109A
##STR00117## 398 399 110A ##STR00118## 522 523 111A ##STR00119##
467 468 112A ##STR00120## 467 468 113A ##STR00121## 481 482 114A
##STR00122## 480 481 115A ##STR00123## 494 495 116A ##STR00124##
425 426 117A ##STR00125## 448 449 118A ##STR00126## 431 432 119A
##STR00127## 419 420 120A ##STR00128## 436 437 121A ##STR00129##
504 505 122A ##STR00130## 506 507 123A ##STR00131## 422 423 124A
##STR00132## 466 467
125A ##STR00133## 478 479 126A ##STR00134## 500 501 127A
##STR00135## 544 545 128A ##STR00136## 530 531 129A ##STR00137##
486 487 130A ##STR00138## 464 465 131A ##STR00139## 508 509 132A
##STR00140## 491 492 133A ##STR00141## 519 520 134A ##STR00142##
494 495 135A ##STR00143## 477 478 136A ##STR00144## 505 506 137A
##STR00145## 466 467 138A ##STR00146## 335 336 139A ##STR00147##
477 478 1404A ##STR00148## 461 462 141A ##STR00149## 489 490 142A
##STR00150## 461 462 143A ##STR00151## 461 462 144A ##STR00152##
461 462 145A ##STR00153## 475 476 146A ##STR00154## 489 490
Other compounds of the invention may be similarly prepared using
methods well known to a person of ordinary skill in the art having
regard to that skill and this disclosure.
IN VITRO EXAMPLES
The following examples illustrate the inhibition of cancer-related
kinases (Aurora A and B and VEGFR2 kinases) by compounds of the
first aspect of this invention and the cytotoxic/cytostatic effect
of the compounds against human cancer cell lines in vitro. These
results are considered predictive of efficacy in human cancer
chemotherapy, as other anticancer agents tested in these assays
have shown anticancer activity in humans.
The cell lines HL60 (human promyelocytic leukemia) and HCT116
(human colon carcinoma) were obtained from the American Type
Culture Collection, Manassas, Va., U.S.A., and HUVEC-2 (human
umbilical vein endothelial cells) from BD Biosciences, Bedford,
Mass., U.S.A. Aurora A kinase was obtained from Upstate Biotech,
Billerica, Mass., U.S.A.; Aurora B kinase from BPS Bioscience, San
Diego, Calif., U.S.A.; and VEGFR2 kinase from Cell Signaling
Technology, Danvers, Mass., U.S.A. The IMAP FP Screening Express
kit was obtained from Molecular Devices, Sunnyvale, Calif., U.S.A.;
the CellTiter-Glo assay kit from Promega Corporation, Madison,
Wis., U.S.A.; and the Cellular DNA Cytometric Analysis Reagent Kit
and BrdU labeling and detection kit from Roche Diagnostics
Corporation, Indianapolis, Ind., U.S.A. All products were used in
accordance with manufacturer's directions. Kinase inhibition assays
and the histone phosphorylation (p-H3) assay were conducted in
duplicate, and the cytotoxicity and HUVEC proliferation assays were
conducted in triplicate, in each case with solvent control.
In Vitro Example 1
Aurora A and B Kinase Assays
Aurora A kinase assay: the test compounds were diluted in
dimethylsulfoxide (DMSO, eight concentrations with serial 3-fold
dilutions) and incubated (final DMSO concentration 2.5%) with
Aurora A kinase at room temperature for 5 min in kinase reaction
buffer with 1 mM dithiothreitol (IMAP FP Screening Express). The
kinase reaction was initiated by adding 100 nM fluorescein labeled
PKAtide and 10 .mu.M adenosine triphosphate (ATP), and allowed to
continue for 45 min at room temperature, after which it was stopped
by adding binding reagent (1:400 dilution in binding buffer A).
After 30 min of room temperature incubation with shaking, the
fluorescence polarization was measured on a LJL plate reader.
Aurora B kinase assay: performed as for the Aurora A kinase assay,
but using Aurora B kinase instead of Aurora A kinase.
In Vitro Example 2
VEGFR2 Kinase Assay
This assay was performed as for the Aurora A kinase assay, using
VEGFR2 kinase, CSKtide as the substrate with 6 .mu.M ATP and 1 mM
MnCl.sub.2, a kinase reaction time of 1 hr, and dilution of the
binding reagent 1:1200 in binding buffer A/binding buffer B
(1:1).
In Vitro Example 3
Histone Phosphorylation (p-H3) Assay
Log phase HCT116 cells were seeded at 2.5.times.10.sup.4 cells/well
in a 96-well plate and allowed to attach overnight. The test
compounds were diluted in DMSO (8 concentrations with serial 3-fold
dilutions) and added to the cells (0.5% DMSO final concentration),
and the cells incubated for 4 hr. The cells were then washed three
times with cold phosphate-buffered saline (PBS), and lysis buffer
was added. After 30 min shaking at 4.degree. C. and centrifugation,
the supernatants were transferred to a nitrocellulose membrane by a
"dot-blot" apparatus. After washing the wells, the membrane was
processed for Western blot. Detection of p-H3 and .beta.-actin was
performed on the same membrane with primary rabbit anti-p-H3 and
mouse anti-.beta.-actin antibodies followed by secondary goat
anti-rabbit IRDye800 and goat anti-mouse AlexaFluor 680 antibodies.
The membranes were scanned on an Odyssey scanner.
In Vitro Example 4
HL60 and HCT116 Cytotoxicity Assays
Log-phase cells were trypsinized, collected by centrifugation, and
resuspended in a small volume of fresh medium, and the density of
viable cells was determined following Trypan Blue staining. Cells
were diluted in fresh media (1.times.10.sup.4 cells/mL for HL60 and
4.times.10.sup.4 cells/mL for HCT116 cells), the test compounds
(concentrations between 0.1 .mu.M and 200 .mu.M, dissolved in DMSO,
50 .mu.L) added immediately after dilution to achieve a final DMSO
concentration of 0.5%, then the suspensions added at 150 .mu.L/well
to 96-well plates, and incubated overnight to allow attachment in
the case of adherent cells. The cells were cultured for three days
(about three doubling times). The cells were then collected by
centrifugation, and 100 .mu.L of the culture supernatant was
replaced by the CellTiter-Glo reagent. After incubation for 15
minutes at room temperature, the plate was read with a
luminometer.
In Vitro Example 5
HUVEC VEGF-Dependent Proliferation Assay
HUVEC-2 cells were seeded at 10.sup.4 cells/well in a 96-well plate
coated with 0.1% gelatin in complete medium. After incubation at
37.degree. C. overnight, the cells were washed twice with PBS,
Medium 199 with 0.1% fetal bovine serum was added, and the cells
were incubated for one day. The compounds were serially diluted in
DMSO, and added to the cells at 0.5% DMSO final concentration.
After 2 hr, VEGF (25 ng/mL) was added; and after a further day,
5-bromo-2-deoxyuridine (BrdU, 10 .mu.M) was added to label
proliferating cells, and the cells incubated for another day. The
plates were then processed using the Roche BrdU labeling and
detection kit, and the VEGF-dependent antiproliferative activity of
the compounds determined.
Compounds of formula A showed the following activity in these in
vitro assays. All numbers are rounded to 1 significant figure;
numbers ">x" indicate that the result was greater than the
maximum limit of quantitation of the assay.
TABLE-US-00002 Aur-A Aur-B VEGFR2 p-H3 HCT116 HL60 HUVEC Cpd.
IC.sub.50, nM IC.sub.50, nM IC.sub.50, nM IC.sub.50, .mu.M
IC.sub.50, .mu.M IC.sub.50, .mu.M IC.sub.50, .mu.M 1A 10 5 10 0.2
0.3 1 0.07 2A 30 6 90 2 2 3 0.05 3A 20 10 90 20 3 4A 4 10 30 >40
2 5A 20 8 20 3 10 >10 6A 20 10 200 2 4 2 7A 7 20 30 >5 6 0.08
8A 10 8 60 1 2 0.2 9A 10 7 60 >5 >20 >20 10A 10 10 40 5 1
2 11A 40 10 >5 4 3 12A 7 6 40 0.1 0.4 0.2 13A 10 3 0.8 0.7 1 14A
50 30 80 >50 8 15A 80 30 40 2 2 16A 100 20 300 2 2 2 17A 300 30
1 0.6 5 5 18A 200 40 500 2 20 19A 400 30 >50 20 20A 90 20 200
>5 8 0.6 21A 200 100 1000 20 22A 80 10 200 2 10 23A 70 6 100 8
50 24A 900 1000 400 >50 25A 200 100 400 >50 10 26A 200 100
1000 >50 20 27A 3 9 10 1 7 28A 5 5 10 4 4 0.04 29A 30 10 100
>5 0.2 30A 10 10 8 1 31A 4 9 3 0.1 0.2 1 32A 10 10 40 0.4 5 3
33A 2 10 9 0.5 0.3 0.3 34A 5 6 10 0.4 0.6 0.2 35A 9 5 20 0.6 0.2
36A 30 40 200 >5 4 37A 30 20 400 0.5 2 4 38A 40 10 200 0.3 0.8 1
0.1 39A 10 5 7 0.9 0.3 0.5 0.06 40A 3 7 3 0.5 0.2 7 0.03 41A 6 4 4
0.1 0.7 0.1 0.2 42A 10 6 20 0.3 0.5 0.03 0.04 43A 6 10 44A 10 10 10
0.2 0.2 0.08 45A 20 7 20 0.9 0.5 0.1 0.01 46A 6 10 40 0.2 0.4 0.2
47A 4 6 20 0.2 0.3 0.3 0.004 48A 10 1 10 0.3 0.6 0.1 0.04 49A 3 5
0.1 0.4 1 50A 20 10 20 0.4 0.3 0.09 51A 7 6 20 0.3 0.5 0.05 0.03
52A 60 60 100 >50 1 53A 3 10 20 0.2 0.3 0.2 0.03 54A 6 10 10 0.4
0.4 0.3 0.03 55A 20 20 20 0.7 0.3 0.05 56A 20 20 20 0.7 0.04 0.04
57A 20 20 10 0.1 0.1 0.02 58A 9 5 9 0.5 0.4 0.06 59A 8 20 6 0.09
0.4 0.05 60A 10 7 5 0.2 0.2 0.08 61A 20 10 4 0.03 0.1 0.2 0.02 62A
70 10 6 0.4 0.06 0.06 63A 30 30 5 0.4 0.2 0.06 64A 50 20 10 0.9 2
65A 30 20 7 0.4 0.2 66A 20 10 2 0.2 0.2 0.05 67A 10 40 4 0.04 0.2
0.02 68A 10 10 3 0.06 0.1 0.06 69A 20 30 2 0.1 0.09 0.02 70A 60 10
4 1 0.6 71A 20 40 4 0.2 0.2 0.09 72A 20 20 20 0.3 0.2 0.08 73A 10 5
6 0.1 0.2 0.2 0.1 74A 20 10 20 0.2 0.3 0.04 0.08 75A 60 10 50 0.4 1
0.2 76A 50 5 80 0.2 0.7 0.1 77A 8 7 9 0.1 0.2 0.3 0.09 78A 60 40 50
0.6 2 1 0.4 79A 20 20 10 0.4 0.1 0.3 0.04 80A 10 4 50 0.09 0.5 0.1
81A 6 7 9 0.09 0.3 0.2 0.08 82A 8 7 10 0.4 0.08 0.03 83A 6 10 60
0.2 0.2 0.2 84A 7 9 20 0.1 0.2 0.1 0.06 85A 5 4 20 0.05 0.3 0.06
0.02 86A 4 8 30 0.2 0.4 0.8 0.03 87A 8 9 10 0.2 0.2 0.1 0.09 88A 4
8 20 0.06 0.3 0.1 0.3 89A 5 5 6 0.1 0.5 0.7 0.08 90A 4 9 4 0.07 0.2
0.1 0.09 91A 2 20 10 0.1 0.3 0.04 92A 70 30 400 0.5 5 0.06 93A 20
20 10 0.1 0.3 0.03 94A 7 7 10 0.08 0.2 0.1 0.05 95A 20 4 20 0.05 2
2 0.05 96A 2 3 6 0.04 0.3 0.1 97A 20 7 0.1 0.6 1 98A 6 8 99A 20 7
100 1 3 2 100A 5 9 10 0.3 0.4 0.09 0.02 101A 7 9 20 1 1 2 0.05 102A
4 7 0.05 103A 20 10 20 2 2 0.1 0.1 104A 30 10 60 2 3 1 105A 5 7 0.1
106A 3 20 30 0.9 2 0.08 107A 8 20 40 >5 5 1 108A 10 10 60 3 2 2
109A 20 9 50 >5 3 0.5 110A 50 20 20 0.2 0.2 0.8 0.08 111A 20 9
100 0.4 2 0.3 112A 20 20 200 0.9 2 0.2 113A 30 20 40 0.4 0.6 0.2
114A 40 20 40 0.05 0.6 0.04 115A 40 30 20 0.2 0.1 0.1 116A 40 8 9
0.08 0.4 0.1 117A 10 8 20 0.3 0.8 0.06 118A 40 10 100 4 2 0.3 0.2
119A 9 6 6 0.2 1 0.2 120A 30 9 2 0.5 2 2 121A 30 8 20 0.09 1 0.9
122A 20 9 20 0.4 4 7 123A 100 40 90 2 4 124A 30 30 100 >20 3
125A 30 10 20 0.2 3 0.6 126A 20 20 70 2 2 0.03 127A 20 6 20 0.2 0.6
0.5 0.04 128A 10 20 60 0.6 0.9 0.06 129A 30 10 100 1 1 0.3 130A 100
9 20 0.3 2 0.4 131A 90 60 200 0.9 2 0.8 132A 60 20 100 2 1 0.6 133A
40 10 60 0.3 0.8 0.2 134A 50 10 80 0.4 4 0.6 135A 30 20 100 0.5 4
0.6 136A 20 30 0.2 0.8 0.2 0.2 137A 80 30 40 2 2 138A 8 8 40 0.5 1
0.4 139A 20 2 30 0.09 0.03 0.04 140A 6 9 20 0.4 0.4 0.3 0.02 141A 4
6 40 0.3 0.9 1 0.02 142A 2 4 10 0.3 0.3 0.2 0.09 143A 4 6 30 0.2
0.3 0.3 0.08 144A 5 6 0.1 0.4 0.5 145A 20 4 10 0.08 0.2 0.4 146A 20
5 0.2 0.4 0.8
In Vitro Example 6
Cell Cycle Analysis in HL60 and HCT116 Cells
Log-phase cells were seeded in a 75-mL flask overnight to allow
cell attachment, with the seeding density chosen so that the cell
culture would be less than 80% confluent on the day of harvest. The
test compounds were added (dissolved in DMSO) at about IC.sub.80 to
achieve a final DMSO concentration of 0.1%, and the cells then
incubated further for one, two, or three days. Following
incubation, the cells were harvested, washed with cold PBS, fixed
in 75% aqueous ethanol, and stored at -20.degree. C. until further
analysis. To determine the cellular DNA content, which reflects the
cell cycle status, the fixed cells were washed twice with
phosphate-buffered saline and then treated with RNase for 30
minutes at 37.degree. C. They were then stained with propidium
iodide, followed by FACS analysis on a Becton Dickinson FACSCalibur
system. All compounds tested induced polyploidy followed by
apoptosis in HL60 cells, and polyploidy in HCT116 cells, indicative
of Aurora kinase B inhibition.
IN VIVO EXAMPLES
In Vivo Example 1
HL60 Xenograft Assay, Oral Administration
Male athymic nu/nu mice, 6-8 weeks old (about 20 g), were implanted
subcutaneously in the right fore flank with about 1.times.10.sup.7
cells of the HL60 (human promyelocytic leukemia) line that had been
grown in antibiotic-free medium for at least two passages. About 6
days after tumor implantation, when the tumor weight was about
50-250 mg, the mice were assigned to treatment groups. Test
compounds were solubilized at 15 mg/mL in 25 wt. % aqueous
hydroxypropyl 3-cyclodextrin. Groups of mice were treated with
compounds 39A, 41A, 75A, 77A, 85A, 95A, and 101A at 150 mg/Kg by
gavage once/day on days 1-5 and 8-10 from the start of treatment,
with vehicle control. Tumor growth inhibition was measured 1-2 days
after the last day of treatment. All compounds tested were active
in this assay, with compound 39A causing 47% inhibition of tumor
growth compared to vehicle, compound 41A causing 81% inhibition,
compound 75A causing 72% inhibition, compound 77A causing 75%
inhibition, compound 85A causing 45% inhibition, compound 95A
causing 59% inhibition, and compound 101A causing 38% inhibition.
Similar studies with compounds 139A, 141A, 142A, and 143A dissolved
at 4, 10, 10 and 10 mg/mL in 0.1M sodium acetate at pH5, dosing at
40, 100, 100, and 100 mg/mL, showed tumor inhibition of 65%, 49%,
89% and 65% relative to vehicle.
In Vivo Example 2
HCT116 Xenograft Assay, Oral Administration
Male athymic nu/nu mice, 6-8 weeks old (about 20 g), were implanted
subcutaneously in the right fore flank with about 1.times.10.sup.7
cells of the HCT116 (human colon carcinoma) line that had been
grown in antibiotic-free medium for at least two passages. About
14-21 days after tumor transplantation, when the tumor weight was
about 50-250 mg, the mice were assigned to treatment groups. Test
compounds were solubilized at 15 mg/mL in 25 wt. % aqueous
hydroxypropyl 3-cyclodextrin. Groups of mice were treated with
compounds 39A, 40A, 60A, 75A, 95A, and 117A at 150 mg/Kg by gavage
once/day on days 1-5 and 8-12 from the start of treatment, with
vehicle control. Tumor growth inhibition was measured 9 days after
the last day of treatment. All compounds tested were active in this
assay, with compound 39A causing 56% inhibition of tumor growth
compared to vehicle, compound 40A causing 18% inhibition, compound
60A causing 37% inhibition, compound 75A causing 60% inhibition,
compound 95A causing 61% inhibition, and compound 117A causing 42%
inhibition. A similar study with compound 139A dissolved at 4 mg/mL
in 0.1M sodium acetate at pH5, dosing at 40 mg/mL, showed tumor
inhibition of 46% relative to vehicle.
FORMULATION EXAMPLES
Formulation Example 1
Formulation for Oral Administration
A solid formulation for oral administration is prepared by
combining the following:
TABLE-US-00003 Compound of this invention 25.0% w/w Magnesium
stearate 0.5% w/w Starch 2.0% w/w Hydroxypropylmethylcellulose 1.0%
w/w Microcrystalline cellulose 71.5% w/w
and the mixture is compressed to form tablets or filled into hard
gelatin capsules containing, for example, 100 mg of the compound of
this invention. Tablets may be coated, if desired, by applying a
suspension of a film-forming agent (for example,
hydroxypropylmethylcellulose), pigment (for example, titanium
dioxide), and plasticizer (for example, diethyl phthalate), and
drying the film by evaporation of the solvent.
Formulation Example 2
Formulation for IV Administration
A formulation for IV administration is prepared by dissolving a
compound of this invention, for example as a pharmaceutically
acceptable salt, to a concentration of 1% w/v in phosphate-buffered
saline; and the solution is sterilized, for example by sterile
filtration, and sealed in sterile containers containing, for
example, 100 mg of a compound of this invention.
Alternatively, a lyophilized formulation is prepared by dissolving
a compound of this invention, again for example as a
pharmaceutically acceptable salt, in a suitable buffer, for example
the phosphate buffer of the phosphate-buffered saline mentioned
above, sterilizing the solution and dispensing it into suitable
sterile vials, lyophilizing the solution to remove the water, and
sealing the vials. The lyophilized formulation is reconstituted by
the addition of sterile water, and the reconstituted solution may
be further diluted for administration with a solution such as 0.9%
sodium chloride intravenous infusion or 5% dextrose intravenous
infusion.
While this invention has been described in conjunction with
specific embodiments and examples, it will be apparent to a person
of ordinary skill in the art, having regard to that skill and this
disclosure, that equivalents of the specifically disclosed
materials and methods will also be applicable to this invention;
and such equivalents are intended to be included within the
following claims.
* * * * *